Tilapia Research Institute of Aquaculture

Professor Brendan McAndrew

CEFAS Tilapia Workshop18th June 2009

Introduction

There has been research on tilapia undertaken at the IoA since 1978

Much of this early work funded by UK overseas development funds.

Wide range of subjects studied -genetics, nutrition, disease, reproductive biology.

Stirling strains widely used by industry

Background Tilapia species gathered from wild in Africa. All collections checked using morphological as

well as genetic techniques to ensure purity. Early work repeated existing studies to obtain

baseline results on known genetic material –hybridisation both intentional and unintentional was widespread in commercial strains making identification difficult.

Single sex tilapia has been an ongoing research topic.

MIXED SEX V’S MONOSEX TILAPIA

Mixed SexTilapia All Male Tilapia

TILAPIAS (Oreochromis spp.)

• Monosex male culture offers a solution to reproduction before harvest: this has been achieved by hormonal masculinisation or through genetic techniques

• Sex determination appears to be largely genetic and monofactorial below about 34oC, but differs between species in the genus (O. niloticus and O. mossambicusXX/XY, O. aureus WZ/ZZ). YY males viable.

• Above about 34oC, temperature affects sex ratio, largely through masculinisation of genetic females

• No identification of sex chromosomes or sex-linked markers until recently - being developed at IOA

Manipulation of sex-ratios in tilapia• Hand sexing, 30g+ fish sexual dimorphism

• Hybridisation, Widely abused, niche use.

• Hormones

•Hormonal sterilisation- unacceptable today.

•Direct - larvae/fry are treated with steroid hormones during sexual differentiation to change sex ratio.

•Indirect - sex determination system is manipulated in broodstock to result in progeny which are all genetically the same sex.

• Temperature dependent sex-determination. 34-38 C can change phenotypic sex. female-male

Hormone sex-reversal Exogenous hormone swamps natural

hormone changes that cause sexual development.

Phenotypic change of sex the neomales or neofemales produced are still the same genetic sex.

Simple highly efficient technique small amounts of hormone applied for labile period.

EU regulation does not allow direct application in human food chain.

According to EU Directive 96/22/EC (entry into force 23 May 1996),

• Contamination from substances with hormonal action and other substances. According to EU Directive 96/22/EC (entry into force 23 May 1996), Member States shall prohibit: (a) the placing on the market of stilbenes, stilbene derivatives, their salts and esters and thyrostatic substances for administering to animals of all species and (b) the placing on the market of betaagonists for administering to animals, the flesh and products of which are intended for human consumption.

They shall, also, prohibit (i) the administering to a farm oraquaculture animal of substances having a thyrostatic, androgenicor gestagenic action and of betaagonists, (ii) the holding of animalson a farm, the placing on the market or slaughter for humanconsumption of farm animals or of aquaculture animals whichcontain the substances referred or in which the presence of suchsubstances has been established, (iii) the placing on the market forhuman consumption of aquaculture animals to which substanceshave been administrated and of processed products derived fromsuch animals,

HORMONAL SEX REVERSAL

F H YSR SD

LABILE PERIOD

DELIVERYHORMONESTART TIMEDURATIONCONCENTRATIONCOMPETITIONNATURAL FOOD

HIGH RATE OF SEX REVERSALHIGH SURVIVAL RATE

F = Fertilisation; H = hatch; YSR = yolk sac resorption; SD = sexual differentiation

Labile period will vary depending on species 10 days for tilapia 100 days for trout and seabass

Direct treatment

Dose between 30-60ppm 17- α Methyltestosterone

(MT) Dose will depend on wide range of

parameters but must be started before 10 days post hatch, swim-up stage.

This require hatcheries to have tight control over fry collection usually egg-robbing and artificial incubation to get the best % reversal.

Indirect hormone treatment This technique is normally used to generate a

specific sex determination genotype. In tilapia we want an all-male system in a

heterogametic species. E.g. XY male XX female. We need to develop YY males or ZZ females. In fish there are several ways to achieve this

result depending on the levels of sophistication available.

Hormone never used in the production fish.

Genetic all-male production in an XX/XY species – Nile tilapiausing hormone treatments

(after Mair et al, 1991)Process involves several labour intensive progeny testing stages.

Chromosome set manipulationInduction of gynogenesis in fish

oogoniagenome duplication replication

1st meiosis

2nd meiosis 1st mitosis

2n

n

2novulation

Fertilise with UV irradiated sperm

1st polar body

2nd pb

50%

100%

YY male O. niloticus :Mitotic Gynogenesis

XX female XY male

DES

MITOTIC GYNOGENESIS

XX females

XY neofemale

YY males

XX female

Progen

F0

F1

F2

Progeny testing will identify neofemales

Mixed XX females and YY males.

FRESH SPERM

YY male production :

Androgenesis

Haploid embryos

Late shock1st mitotic division

Partial pedigree of androgenetic male O.niloticus and the % males in progenywhen crossed to normal females.

All-male Stirling red tilapia

Developed from pure Egyptian O.niloticus.

Dominant red gene- no melanophores in the epidermis.

Pure breeding strains available, widely distributed.

Androgenesis used to produce YY males and can be supplied to generate all-male fry in Stirling strain.

This is the latest generation of Stirling red tilapia YY male

Chromosome set manipulations

Offer rapid way to generate new genotypes such as YY males.

Useful technology to study the inheritance of sex-determination mechanisms and other complex traits.

Useful technology for gene mapping. Triploidy- not yet commercial reality.

Temperature sex-determination

Evidence that sex-ratio can be biased towards males by raising individuals from susceptible families at +34 C.

Selection for lines that produce a higher male % has shown improvements upto 90% male.

Evidence from high %male lines that high temperature can reduce this %.

Is this line worth pursuing?

Reproductive biology of tilapiaHatchery production of tilapia fry relatively inefficient

-low fecundity

-asynchronous spawning

-need large numbers of females

-hormonal control ofreproduction has not worked

-evidence that light is a majorcue and that tilapia respond to day length and intensity

Photoperiod experiments Female Nile tilapia from same family

ongrown under identical conditions to maturity.

Separated into four different light regimes 6D:18L, 12D:12L, 18D:6L and 24L.

Females maintained on these regimes for 6 months and spawning activity monitored.

All eggs counted and measured.

Photoperiod control of reproduction in tilapia

Number of Spawns

0

20

40

60

80

100

6L:18D 12L:12D 18L:6D 24L

Spa

wns

Total per month

Egg production

0

10000

20000

30000

40000

50000

Sep-01 Oct-01 Nov-01 Dic-01 Ene-02 Feb-02

Egg

s

6L:18D 12L12D 18L:6D 24L

Inter-spawning-interval

0

5

10

15

20

25

6L:18D 12L:12D 18L:6D 24L

days

ab b

cac

Extended day lengths (18,24hr) increased spawning activity –reduced Inter Spawning Interval (ISI).

Highest and most consistent egg product in 18hr day

(Campos-Mendoza et al 2004)

PhotoperiodFecundity

012345678

6L:18D 12L:12D 18L:6D 24L

Numb

er of

eggs

Fecundity (x1000) Relative fecundity (egg/g)

ab b

b

b b a a

y = 0.1517x + 0.1938R2 = 0.3202; p 0.000

y = 0.4405x + 0.2616R2 = 0.3539; p 0.000

0

0.2

0.4

0.6

0.8

1

1.2

1 1.2 1.4 1.6 1.8 2

Log10 ISI

Log 1

0 mm

Diameter mm Volume mm3

Longer days increased relative and total fecundity

Shorter ISI resulted in more but smaller eggs

(Campos-Mendoza et al 2004)

Potential for photoperiod control 18L:6D produced 58% more eggs than the

ambient 12L:12D photoperiod. Fish under 18L:6D significantly higher

total and relative fecundity, reduced ISI and greater clutch size.

Some photoperiod better than continuous light – entrain rhythm.

Further work on mechanism underway. Evidence that they are very sensitive to

light.

Light Intensity - growth

Recent work has shown that growth performance can be improved by using continuous medium to low lighting regimes.

Up to 20% improvement in weight at 118 dph under experimental conditions needs to be repeated under commercial conditions.

In other species benefits not seen until later growth stages.

Table 1. Light intensities in Watts m-2 and Lux (mean SE) measured at the bottom and surface of the tanks for each

experimental treatment during day time.

Treatment Watts m-2 Lux

LL High topbottom

3.0 0.2/4.6 06

684.0 32.0/1031.0 104.0

LL Medium 0.5 0.1 / 0.7 0.1

141.5 17.5/172.5 10.5

LL Low 0.04 0.0/

0.0 0.0

4.5 0.5/ 8.0 1.0

Control 0.7 0.1 / 0.9 0.2

172.5 22.5/190.5 30.5

Weight over time in Nile tilapia raised up to 118 days post hatch under different light intensities (High LL, Medium LL, Low LL and Control 12L:12D). Values are expressed as mean SE (n = 33-75 / replicate). Superscripts indicate significant differences between treatments at a given time point.

Different photoperiod control systems widely used in fish culture in NW Europe to control sexual maturation and improve growth performance in salmon, trout and marine species.

> 30% improvement on growth performance with extended days.

Extended day-length in hatchery likely to improve fry yields.

Extended day-length in ongrowing likely to improve overall growth rate –shorter production cycles.

Genomic techniques being used at the moment to study many of the traits described- new developments to come

New light technology used by cod farmingoperations in Norway and Scotland.

Scientists involved

Dr David Penman Dr Hérve Migaud Dr Jim Myers Dr M. Gulam Hussain Dr Antonio Campos-Mendosa Dr Rafael Campos-Ramos Dr Antonio Mendoza. Dr Chris Martinez.