Rafael Martinez-Garcia, Ulises Hernández-Vidal, Gabriela Arias-Jimenez and Wilfrido M. Contreras-Sánchez

APROACH TO THE RESEARCH IN SEX IDENTIFICATION IN TROPICAL GAR Atractosteus tropicus

Laboratorio de Acuacultura DACBiol, UJAT

Project funded by

Tropical gar value

• Good price and high regional market demand for traditional consumers

• Fry production increasing because of new farms operation (private & rural)

• Broodstock for sale demand increasing: request of sex identification for selection and evaluation

Tropical gar sex identification main problems

• Scarce adult female availability (suitable for brookstock incorporation)

• Sexual dimorphism identification only feasible for oldest or ripe females.

• Anatomic difficulties for sex identification by conventional techniques

Sexual dimorphism

Adult fish handling experience necessary and without precision

Lab produced adults: not applicable because fat accumulation

Plasma vitellogenin

Actual techniques for sex identification

Antibodies obtention anti-VTG

Inmunization:Lab rabbits 600-800 gr.200 g VTG + saline

solution and adyuvant. Inmunizations every two

weeks for three months.Antibodies checkings

Agarose gels (Ouchtherlony, 1961)

Cross tests:Serum of organisms induced

and no induced with estradiol.

Semi purified precipitates and chromatographics fractions

Technique of Plasma Vitellogenin for sex identification

Sampling

Inmunodifusion inInmunodifusion inAgarose GelesAgarose Geles11%% per 24 h. per 24 h.

Centrifuge at 5000 rpm 10 min

+ PMSF

Serum

TESTS FOR SEX IDENTIFICATIONTESTS FOR SEX IDENTIFICATION

(Hernandez,2000)

Cross tests and antibodies selectionCross tests and antibodies selection

Reaction among:Reaction among: anti-VTG serum and purified VTG anti-VTG serum and purified VTG

With EstradiolWith EstradiolWithout EstradiolWithout Estradiol

Reaction among:Reaction among: anti-VTG serum and males plasma anti-VTG serum and males plasma

treated with Etreated with E22

H and EH and E400x400xc : c :

Spermatocistos Spermatocistos e : e :

SpermatozoonSpermatozoon

e

c

c

Sex identification in AdultsSex identification in Adults

MalesMales

(Mendez,2002)

H and EH and E100x100x

n : nucleus n : nucleus zp : zona zp : zona pellucidapellucida

o : ovoplasmao : ovoplasma

FemalesFemales

nn

zpzp

oo

(Mendez,2002)

OBJECTIVEOBJECTIVE

To determine the plasma vitellogenin levels To determine the plasma vitellogenin levels in adult tropical gars, in adult tropical gars, A. tropicus,A. tropicus, during an during an annual cycle.annual cycle.

Annual cycle of plasma vitellogenin in Annual cycle of plasma vitellogenin in adults of tropical garadults of tropical gar

METHOD

(diameter of the immunoprecipitation circle)2 –(diameter of the well)2 Matsubara and Sawano, 1995

Vg QuantificationSimple Radial Inmunodiffusion : SRID (Mancini

et al, 1965)– Agarose gels (Tris:Tricinn:Lactate of Ca)

• serum aVg 1:1000– Standard Curve from 0 a 15 g of Vg.– 3µL of sample per well– Incubation for 48 hrs, press and stain– Relative concentration of Vg

• Matsubara and Sawano (1995)

RESULTS

Isolation of Vg and Isolation of Vg and production of production of aVgaVg

Gel of SDS-PAGE organism treated with Gel of SDS-PAGE organism treated with estradiol and controls. The arrow estradiol and controls. The arrow

indicate the plasma Vg indicate the plasma Vg

Samples showing animals Samples showing animals treated with estradiol and treated with estradiol and samples with the Vg samples with the Vg precipitated precipitated

Production Production of of aVgaVg

Gel of antibody against precipitated Gel of antibody against precipitated Vg of organisms with plasma Vg of organisms with plasma

estradiolestradiol (showing different dilutions (showing different dilutions of aVg)of aVg)

SDS-PAGE gel showing the SDS-PAGE gel showing the similitude between similitude between

Lv and Vg precipitation Lv and Vg precipitation

a b

Isolation of LvIsolation of Lv

Production of Production of aLvaLv

Gel of antibody against Lv of ovocites extract. The maximum Gel of antibody against Lv of ovocites extract. The maximum antigen action was found at dilution of 1/8antigen action was found at dilution of 1/8

Vg quantificationVg quantification

(1) Female with reaction well defined, (2) organism with hardly perceivable levels (males) and (3) organisms with non detectable levels (males).

1 32

Feb Mar Apr May Jun Jul Agu Sep Oct Nov Dec Jan

0

5

10

15

20

25

30

35

Máx

Mín

Avegarge SE

mgV

g m

L-1

Concentration of plasma Vg in A. tropicus during the yearConcentration of plasma Vg in A. tropicus during the year

• In females and males of A. tropicus it is possible detect and quantify plasma Vg using aLv serum obtained from the separation of Lv from ovocites in mature females in this specie.

• The aLv serum reacted positively against male plasma (treated with estradiol) and mature females, this fact confirm that aLv serum can be used for recognizing Vg protein

• A. tropicus showed the maximum Vg levels in April and July (this last value is within the spawning season), this fact give us the idea that the organism were in an active vitellogenic phase

• The presence of the minimum levels in December and February could be indicators that the synthesis of Vg is low, this agrees with the end of the reproductive period.

• The plasma Vg levels could be an indirect indicator of the gonadal activity and the success of hormonal spawning induction can be achieved taking into account the maximum levels observed just before the spawning activity.

CONCLUSIONSCONCLUSIONS

Vitellogenin & Mucus Vg is present in mucus of many fish species (Kishida et al, 1992; Kishida and Speaker 1994, 2000).

Copious mucus production, easy and fast to collect

Tropical gar features…

SEX IDENTIFICATION OF TROPICAL GAR Atractosteus tropicus JUVENILES BY VITELLOGENIN

DETECTION IN SKIN MUCUS

Objetive

• To evaluate if Vg is detectable in tropical gar skin mucus juveniles to obtain a new technique for sex identification and improve the broodstock management

Vg production and detection:

• UJAT produced juveniles were injected weekly with 1mg E2 /kg (treated) or with vehicle (control)

• Blood and Mucus samples were collected each week before a new injection

• Plasma samples were processed by 6% SDS-PAGE and Cross-reactivity for Vg in Calcium lactate buffer agarose gels

• Mucus samples were extracted with PBS-Tween 20 buffer

and added with PMSF

Mucus and blood collection

• dfg

Plasma detection and cross reactivity of Vg

C

T

C

C T

T

T

T

• T: E2 treated• C: Control

TCTC C T

• Plasma Vg was detected from the 1st to 4th week in E2 treated juveniles and absent in control animals.

Mucus detection and cross reactivity of Vg

C C TT C T C T• Mucus “Vg” was detected from the week 3 in E2 treated juveniles and

absent in control animals.• Some difficulties were observed for mucus processing like low solubility

and flotation

• T: E2 treated• C: Control

T TT TC C C C

CONCLUSIONS • Vg is detected in plasma and skin mucus of

tropical gar using SDS-PAGE and a-Vg

• Skin mucus Vg detection could be a new technique for sex identification in fish

• More information and research will be necessary for improve the technique

Funding for this research was provided by theAquaculture

Collaborative Research Support Program,

CONACYT and UJAT

The Aquaculture CRSP is funded in part by United States Agency for International

Development (USAID) Grant No. LAG-G-00-96-90015-00 and by participating institutions.

Acknowledgments

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