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

APROACH TO THE RESEARCH IN SEX IDENTIFICATION IN TROPICAL GAR Atractosteus tropicus. Rafael Martinez-Garcia, Ulises Hernández-Vidal, Gabriela Arias-Jimenez and Wilfrido M. Contreras-Sánchez. Laboratorio de Acuacultura DACBiol, UJAT. Project funded by. Tropical gar value.

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

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  1. APROACH TO THE RESEARCH IN SEX IDENTIFICATION IN TROPICAL GAR Atractosteus tropicus Rafael Martinez-Garcia, Ulises Hernández-Vidal, Gabriela Arias-Jimenez and Wilfrido M. Contreras-Sánchez Laboratorio de Acuacultura DACBiol, UJAT Project funded by

  2. 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

  3. 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

  4. 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

  5. Technique of Plasma Vitellogenin for sex identification 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 Antibodies obtention anti-VTG

  6. Centrifuge at 5000 rpm 10 min + PMSF Serum Inmunodifusion in Agarose Geles 1% per 24 h. Sampling TESTS FOR SEX IDENTIFICATION (Hernandez,2000)

  7. Reaction among: anti-VTG serum and males plasma treated with E2 Without Estradiol With Estradiol Reaction among: anti-VTG serum and purified VTG Cross tests and antibodies selection

  8. c e c Sex identification in Adults Males H and E 400x c : Spermatocistos e : Spermatozoon (Mendez,2002)

  9. o n zp Females H and E 100x n : nucleus zp : zona pellucida o : ovoplasma (Mendez,2002)

  10. 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.

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

  12. 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)

  13. Samples showing animals treated with estradiol and samples with the Vg precipitated RESULTS Isolation of Vg and production of aVg Gel of SDS-PAGE organism treated with estradioland controls. The arrow indicate the plasma Vg

  14. SDS-PAGE gel showing the similitude between Lv and Vg precipitation a b Isolation of Lv Production of aVg Gel of antibody against precipitated Vg of organisms with plasma estradiol(showing different dilutions of aVg)

  15. Production of aLv Vgquantification 1 3 2 Gel of antibody against Lv of ovocites extract. The maximum antigen action was found at dilution of 1/8 (1) Female with reaction well defined, (2) organism with hardly perceivable levels (males) and (3) organisms with non detectable levels (males).

  16. 35 Máx Mín 30 Avegarge SE 25 20 15 10 5 0 Feb Mar Apr May Jun Jul Agu Sep Oct Nov Dec Jan Concentration of plasma Vg in A. tropicus during the year mgVg mL-1

  17. 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. CONCLUSIONS

  18. Tropical gar features… • Copious mucus production, easy and fast to collect 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).

  19. 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

  20. 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

  21. Mucus and blood collection • dfg

  22. C T C C T T T C T C T T • T: E2 treated • C: Control T C 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.

  23. C T C T C T C T • T: E2 treated • C: Control Mucus detection and cross reactivity of Vg • 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 C T C T C T C T

  24. 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

  25. Acknowledgments Laboratorio de Acuacultura-UJAT 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.

  26. Come and join us…. International Network for Lepisosteid Research 150 Millions years being strongs We are not a evolutionary fad We are not primitives, We are persistent http://www.lepisosteidos.ujat.mx/

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