APROACH TO THE RESEARCH IN SEX IDENTIFICATION IN
TROPICAL GAR Atractosteus tropicus
Rafael Martinez-Garcia, Ulises Hernández-Vidal, Gabriela AriasJimenez and Wilfrido M. Contreras-Sánchez
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
Actual techniques for sex identification
 Sexual dimorphism
 Adult fish handling experience necessary and without precision
 Lab produced adults: not applicable because fat accumulation
 Plasma vitellogenin
Technique of Plasma Vitellogenin 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
TESTS FOR SEX IDENTIFICATION
Centrifuge at
5000 rpm 10 min
+ PMSF
Serum
Inmunodifusion in
Agarose Geles
Sampling
1% per 24 h.
(Hernandez,2000)
Cross tests and antibodies selection
Reaction among:
anti-VTG serum and males plasma
treated with E2
Without Estradiol
Reaction among:
anti-VTG serum and purified VTG
With Estradiol
Sex identification in Adults
Males
c
H and E
400x
c : Spermatocistos
e : Spermatozoon
e
c
(Mendez,2002)
Females
o
n
H and E
100x
n : nucleus
zp : zona pellucida
o : ovoplasma
zp
(Mendez,2002)
Annual cycle of plasma vitellogenin in
adults of tropical gar
OBJECTIVE
To determine the plasma vitellogenin levels
in adult tropical gars, A. tropicus, during an
annual cycle.
METHOD
(diameter of the immunoprecipitation circle)2 –(diameter of the well)2
Matsubara and Sawano, 1995
Vg Quantification
Simple 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
production of aVg
Samples showing animals treated with
estradiol and samples with the Vg
precipitated
Gel of SDS-PAGE organism treated with estradiol
and controls. The arrow indicate the plasma Vg
Production
of aVg
Isolation of Lv
SDS-PAGE gel showing the similitude between
Lv and Vg precipitation
a
Gel of antibody against precipitated Vg of
organisms with plasma estradiol (showing
different dilutions of aVg)
b
Production of aLv
Gel of antibody against Lv of ovocites extract. The maximum antigen action was
found at dilution of 1/8
Vg quantification
1
2
3
(1) Female with reaction well defined, (2) organism with hardly perceivable
levels (males) and (3) organisms with non detectable levels (males).
Concentration of plasma Vg in A. tropicus during the year
35
Máx
Mín
30
Avegarge SE
mgVg mL-1
25
20
15
10
5
0
Feb
Mar
Apr
May
Jun
Jul
Agu
Sep
Oct
Nov
Dec
Jan
CONCLUSIONS
•
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.
SEX IDENTIFICATION OF TROPICAL GAR
Atractosteus tropicus JUVENILES BY VITELLOGENIN
DETECTION IN SKIN MUCUS
Vitellogenin & Mucus
 Vg is present in mucus of many fish species (Kishida et al, 1992;
Kishida and Speaker 1994, 2000).
Tropical gar features…
 Copious mucus production, easy and fast to collect
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
• Plasma Vg was detected from the 1st to 4th week in E2 treated
juveniles and absent in control animals.
T
C
T
C T C TC T
T
C
T
T
C
• T: E2 treated
• C: Control
Mucus detection and cross reactivity of Vg
• Mucus “Vg” was detected from the week 3 in E2 treated
C animals.
T C T C T C T
juveniles and absent in control
• Some difficulties were observed for mucus processing like low
solubility and flotation
C T CT C T C T
• T: E2 treated
• C: Control
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
Acknowledgments
Laboratorio de Acuacultura-UJAT
Funding for this research was provided
by the
Aquaculture
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.
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