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
Laboratorio de Acuacultura-UJAT
Come and join us….
http://www.lepisosteidos.ujat.mx/
International Network for Lepisosteid Research
150 Millions years being strongsWe are not a evolutionary fadWe are not primitives, We are persistent