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Reproduction and Ecological Aspects of Brek (Puntius orphoides) and Lukas (P.bramoides) As a Basic Domestication and Aquaculture Diversification

Priyo Susatyo, Sugiharto, W. LestariFaculty of Biology, University of General Sudirman, Purwokerto

Korespondens: Tel.: 085869718260, fax: (0281) 631700E-mail address: p_susatyo@yahoo.com

Abstract

Research on the search efforts on aspects of reproduction, ecological several species of fish catches Brek (Puntius orphoides), and Lukas (P. bramoides) from the river Serayu Banyumas (in an integrated and systematic) as the basis for domestication and diversification efforts of intensive aquaculture. The study was conducted with the aim of: (1) acclimating and pre-domestication of two types of fish caught from the River Serayu Banyumas (Brek and Lukas) in natural ponds (ex situ), (2) know the hormonal profile of estrogen (17ß-estradiol), progesterone, testosterone, FSH male and female fish during the reproductive cycle pre-domestikasi/satu period, (3) know the histological development of the testes and ovaries of fish during the reproductive cycle, (4) identified several aspects of physiological and ecological (habitat characteristics of the new substrate, feeding habits) , (5) Spawning natural (natural spawning in the pond cultivation), assisted by GnRH analogue induction method. Preparation of dosage histiologis with paraffin method, Haematoxylin eosin staining; hormone analysis by ELISA method using a machine Microplate Reader LB-6200-Labotron. Food preference test of media / substrate maintenance (index of electivity and index of preponderance. The result: Fish Brek and Lukas have managed adaptive (preference index data feed / Index of preponderance respectively 56.893 and 62.854. Profile of hormone until the end of the period pre-domestication 8 months, a sequence of Brek and Lukas: (17ß-estradiol = 1058.06 to 854.85 pg. / ml; 395.18 to 854.85 pg / ml), progesterone = 0.29 to 0.72 ng / ml; 0.20 to 0.52 ng / ml); (FSH = 10.87 to 15.68 mIU / ml, 8.26 to 13.74 mIU / ml); (testosterone = 4.18 to 9.92 ng / ml, 3.98 to 9.34 ng / ml). there are 5 (five) stages of oogenesis development of Brek and Lukas were the chromatin nucleolar stage (CNS); perinucleolar stage (ps); cortical alveolar stage (cas); vitellogenic stage ( vs.) and mature / RIPE stage (ms). Stages of spermatogenesis of Brek, and Lukas namely as the stage of spermatogonia, primary spermatocyte, secondary spermatocyte, spermatid, spermatozoa. In the pre-domestication of 4 months only capable of holding Lukas breeding, and at the end of the period pre-domestication of 8 months, Brek and Lukas managed to spawn. All of the parent stock still in a natural pond and will continue to be domesticated to be a super parent stock someday. Post-hatching larvae remain preserved for further research.

Keywords: pre-domestication, Brek fish (Puntius orphoides), Lukas fish (P. bramoides); index elektivitas feed, reproduction

Introduction

Serayu River covers a very wide catchment area, covering the district of Wonosobo, Banjarnegara, Purbalingga, Banyumas and Cilacap has a diversity of fish species that live there and some of Familia Cyprinidae with a number of species and a considerable number of individuals (Lester and Sugiharto, 2008). There are seven species of fish from the Cyprinidae family who successfully caught from along the upstream to the middle part watershed Serayu, namely Osteochilus hasselti CV (Nilem); O. Microcephalus; O. kahajenensis; Puntius javanicus (tawes); P. orphoides (Brek); Rasbora argyrotaenia; R. lateristriata. Meanwhile, according Harsini (2005) and Halamsyah (2000), along the watershed Serayu Banyumas area (middle) successfully captured 9 species of the family Cyprinidae, ie, Puntius javanicus (tawes); P. bramoides (Lukas); P. binotatus (Beunteur); Osteochilus hasselti C.V. (Nilem); O. Microcephalus; O.kahajenensis;

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Rasbora argyrotaenia; R. lateristriata. But Brek and Lukas fish species are now located in a critical condition (number the less). In Banyumas region, some species of freshwater fish that has long been cultivated and have significant economic value to date is the carp, Nilem, Catfish, tawes, fish Mas, Nila, Mujahir (Susatyo and Sugiharto, 2001; Susatyo and Soeminto, 2002) . One effort to address the domestication of some species of fish catches (Brek and Lukas) which tends to decrease the amount is to conduct initial activities pre-domestication with a series of studies in order to gain knowledge and techniques to prepare for the test fish in a condition ready for naturally grown in pond culture (ex- situ). In order to utilize the Brek and Lukas fish in a sustainable manner, it is necessary pre-domestication by referring to the basic biological and ecological data, such as biological aspects of fish reproduction test, both on aspects of anatomy-histology process of gametogenesis, hormonal regulation and reproductive strategies, physiology, behavior / eating habits , the ability to spawn, and substrate characteristics of their habitat in the wild. According to Kime (2008), analogies using information from other fish species to predict aspects of fish reproduction test will not be able to obtain valid information, especially to determine whether species of fish able to test the complete reproductive cycle in nature, can not fully be done considering the high plasticity reproduction in each species of fish.Thus, research should be done directly on the fish Brek and Lukas to obtain information that supports all aspects of the terms of the success of domestication. Hopefully, through this research (either on the field and laboratory scale) can be found domestication model tested and applicable to society as well as the other user. This research activity carried out activities with the aim to: (1) acclimation and pre-domestication of Brek and Lukas fish, fish caught from the River Serayu in natural ponds that meet certain conditions cultivation, (2) knowing that periodic hormonal profile of estrogen (estradiol-17β), progesterone, testosterone, FSH parent male and female fish during the period pre-domestication / a reproductive cycle, (3) know the histological development of the testes and ovaries of fish during the reproductive cycle is evaluated by measuring the IKG and observe the development of oocytes and spermatozoa, (4) know some ecological and physiological aspects (substrate characteristics of new habitat, feeding habits), (5) Spawning natural (natural spawning in pond aquaculture) or assisted with the induction method with ovaprim.

Material and Methods

The study with survey methods. Research materials used in this study are fish and fish-Brek Lukas males - females with body weight 250-400 g (Brek) and 100-200 g (Lukas), the catch from the river along Serayu. Fishing activities conducted in the river that passes through Banyu-mas Serayu course, includes the region of the river begins its flow Serayu between hamlet Congot, sub Kemangkon Purbalingga as "up-stream, Kedung Uter Banyumas District as a middle stream. While the south west region Serayu river is the river after leaving the gate weir Motion Ser-ayu Serayu flow past a location adjacent to the Cilacap district as down stream. Fish catches (Brek and Lukas) acclimating in two ponds maintenance size 10 x 10 m2 which parti-tioned into 16 plots-screen, using bamboo frame.  Adequate water supply to the in let and out let that pass. The experiment was conducted from February 2008 until December 2009. Group stage fish pre-domestication 1 month (March-April 2009), hormonal and their gametogenesis information unearthed in May 2009, pre-domestication 2 months (April-June 2009), dug up the information in June 2009; pre-domestication 4 months (February-June 2009), unearthed information on the end of July 2009; pre-domestication 8 months (February-October 2009), explored the information in October 2009; since the beginning of this maintenance in the form of news feed pelleted artificial feed and alternating with sente leaves and cassava leaves. Measurement of hormone levels performed using the method of EIA / ELISA, the kit's catalog-estradiol EIA kit (for estradiol), progesterone EIA-kit (for progesterone) and testosterone EIA-kit (for testosterone). Before the measurement of hormone levels, calibrated according to procedures specified by the Directive Kit. Assay was performed using a machine Microplate Reader LB-6200-Labotron.  Ovaries and testes removed from the abdominal cavity through surgery. These organs were fixed with a solution of the Normal Buffer Formalin (NBF) for at least 24 hours at room tem-perature. Furthermore, histological preparations were made by paraffin method and stained with Mayer's Haematoxylin-Eosin. Oocytes are grouped into five phases namely chromatin nucleolar

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stage, perinucleolar stage, cortical alveolar stage, vitellogenic (yolk) stage and mature / RIPE stage. The size of the diameter of oocytes at each stage of development in each ovary was ob-served to identify the type of this phase. (Çakici and Üçüncü, 2007). For premises stages of sper-matogenesis spermatogonia, primary spermatocytes, secondary spermatocytes and spermatozoa

(Brito and Bazzoli, 2004).  Induction of natural spawning by injecting / induce GnRH analogue in male and female master of ostensibly mature sex, using ovaprim 0.5 cc / kg. Further analysis of physical and chemi-cal water sampling conducted at the station Serayu river and pond experiments including tempera-ture, pH value, dissolved O2 and CO2 content of the free.Tabulation and Data Presentation. Calculation of gonado-somatic index (GSI) or Gonad Maturity Index (IKG). IKG is calculated with the formula = gonad weight (gonad weight + weight) x 100%. The data in the form of levels of each steroid hormone and gonadotropin (Tables 2 and 3), IKG (Table 4), also index of electivty and index of preponderance (Table 6) are presented in the form of tables and bar charts. Male and female gonads histologically analyzed descriptively. 

Results and Conclussion

Based on observations during the research activities following results are obtained test fishing locations can be seen in Table 1.

Table 1. Observations on Three Station Locations Fishing in River Flow Serayu

No Village Name Altitude South Latitude East Longitude1 Congot (upstream) 384 m dpl 070 20’ 499’’ 1090 20’ 796’’2 Kedung Uter (middle stream) 285 m dpl 070 30’ 519’’ 1090 17’ 422’’3 Bendung Gerak (down stream) 254 m dpl 070 31’ 393’’ 1090 12’ 139’’

Table 2. The mean levels of steroid hormones in fish Brek at the end of the period pre-domestication 1, 2, 4 and 8 months

Sex of fish

HormoneThe mean levels of hormones (data ± SD)

1 month 2 months 4 months 8 monthsfemale Est (pg/ml) 1293.59 ± 45.44 1021.63 ± 27.56 1541.08 ± 72.52 1493.27 ± 183.09

Prog (ng/ml) 0.31 ± 0.02 0.51 ± 0.03 0.47 ± 0.19 0.84 ± 0.03

FSH (mIU/ml) 10.75 ± 0.10 13.64 ± 0.62 13.82 ± 1.08 14.09 ± 1.45

male Testos(ng/ml) 5.07 ± 0.61 7.50 ± 0.70 9.10 ± 0.17 8.95 ± 0.87

Table 3. The mean levels of steroid hormones in fish Lukas at the end of the period pre-domestication 1, 2, 4 and 8 months

Sex of fish

HormoneThe mean levels of hormones (data ± SD

1 month 2 months 4 months 8 months

female Est (pg/ml) 652.77 ± 17.36 489.00 ± 15.29 455.94 ± 37.40 750.22 ± 86.43Prog (ng/ml) 0.26 ± 0.03 0.42 ± 006 0.47 ± 0.04 0.69 ± 006FSH (mIU/ml) 8.97 ± 0.76 11.46 ± 1.047 10.72 ± 1.90 11.89 ± 1.72

male Testos(ng/ml) 4.38 ± 0.33 5.88 ± 0.64 6.88 ± 0.06 8.66 ± 075Description :

Est = estradiol; Prog = progesterone; Testos = testosterone; FSH =Follicle Stimulating Hormone

Gametogenesis Fish Test. Lukas and Brek testes have the same shape and formation, single and two-

pronged, clear transparent aspect. Ovary of Brek and Lukas as well as ovarian Cyprinideae others will be paired organs fused at the anterior, situated to the anterior organ of viscera with a range of bright yellow, dark yellow and orange yellow accordance genital maturity.Activities oogenesis. Based on histological observation of gonadal female test fish, both Brek, and Lukas, both showing a picture oocyte development level is relatively similar. All stages of oocyte development in both types of test fish at each end of each period pre-domestication found, only differ in the proportions. Histologic evaluation of

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the female gonads showed that there are five stages of oocyte development, namely chromatin nucleolar stage (CNS), perinucleolar stage (ps), cortical alveolar formation stage (cas), vitellogenic stage (vs) and mature / RIPE stage (ms) (Figure 3 .) Stages of oocyte development of two types of test fish (Brek and Lukas), although relatively similar, but differ in oocyte diameter at each stage of development (Table 4).

Gonad Maturity Index. IKG value all three types of female fish showed an upward trend at the end of each period pre-domestication (Figure 1).

Figure 1. Gonad Maturity Index of Brek and Lukas, female (left) and male (right) at the beginning and end of each period pre-domestication

Figure 2. The proportion of oocytes at stage oogenesis Brek Parent (left) and Lukas (right)   Description:     P1b = pre-domestication 1 month; P2b = pre-domestication 2 months; P4b = pre-domestication 4 months; P8b = pre-domestication 8 months; (CNS = nuclear chromatin stage; ps = perinuclear stage; ca = cortical alveolar stage; vs = vitellogenic stage; ms = mature / RIPE stage)

Table 4. The variation of the diameter of oocyte developmental stages in each type of test fish

No Type of oocyte stagesDiameter of oocyte (µm)

Brek Lukas 1 chromatin nucleolar stage (cns) 129 – 137 202 - 237 2 perinucleolar stage (ps) 146 – 219 285 - 380 3 cortical alveolar stage (cas) 286 – 302 332 - 366 4 vitellogenic stage (vs) 396 – 428 446 - 503 5 mature/ripe stage (ms) 541 – 646 684 - 788

Activities of spermatogenesis. Identification of testicular histology showed that the microscopic structure of both types of

fish testis good test Brek and Lukas, all of the family Cyprinidae is the same, ie, including the type of testicular lobuler berlumina.Testicle Brek and Lukas totaled a pair, or single-like forked, this organ elongated, hanging on the wall of the body by the lining of the dorsal mesentery. Testicle beraspek transparent, clear and in this organ anatomy covered by the body fats. Testes are

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organized as a anastomosa / webbing of lobule-lobule which met in the main channels namely ductus spermaticus sperm. Two zones can clearly be seen in the observations on testis cross section. In the outer region is dominated by a structure with walls of seminiferous lobules contain epithelial cells germinalis spermatogenic. Region next to the lumen-lumen composed of lobules that are filled by spermatozoa. Type of cells from the testes in the cooking sequence in accordance with the endorsement of a screening test for endocrine active substances in fish, the OECD in Brito and Bazzoli, 2004) are: (1) spermatogonia: the largest cell type and consist of vesicular nuclei with nuclear membrane and firm nukleoli , (2) Spermatocyte: primary spermatocyte secondary spermatocyte is greater than, (3) spermatid: the smallest cell type with solid core and a narrow circle in which asidofilik cytoplasm, (4) Spermatozoa: mature cells with round nuclei and dark beraspek berflagella. Spermatogenic cells are scattered randomly in the testes in groups of cell types. In the lobule lumina there are several groups of cells residing in the same spermatogenic stage. Sertoli cells are also found, located on the wall lobula with other germ cells.In the period pre-domestication 1 month and 2 months, the testes of fish Brek and Lukas are at initial stages of ripening, the small amount of spermatogonia, primary spermatocytes and secondary spermatocytes in relatively large quantities, but the number of spermatozoa stage a little. While at the end of the period pre-domestication 4 and 8 months, has reached mature stage / RIPE stage, partly lobula lumina dominated by spermatozoa with relatively high amounts (Figure 4).

Oocyte developmental stage Description of histological structure

Chromatin nuclear stage Oocytes are spherical cells / small ball containing a nucleus located in the central cell.Nucleus containing one to four nucleoli together with webbing khromatin. Cytoplasm is a thin layer, and are strongly basophilic (like on the coloration of its base). Tues follicular difficult to be observed ( ) = 30µ

Perinuclear stage Total of nukleoli increased and arranged along the underside of the nuclear membrane.Large nucleus and cytoplasm is surrounded by the increase in mass that looks look less basophilic. Follicular cells is the monolayer, composed of a single layer of flattened cells cover-ing the oocyte ( ) = 30 µCortical alveoli stage Vesicle-filled cytoplasm appearance of light vesicles (cortical alveoli). Vesicles begin to accumulate on the edge / peripheral oocyte. Nucleus remains perinukleoler. The nuclear membrane starts to become tangled. One pack is thin primary asidofilik, radiata zone visible at the edge of the nucleus. Follicular layer visible at the edge of the oocyte ( ) = 30 µ

Vitelogenic (yolk) stage ocyte size increases. Small yolk granules visible as a ring-shaped area is asidofilik in the cytoplasm. The nucleus is still tangled. Zona radiata looks bright / bright as a non-cellular bands are very asidofi-lik. Follicular layers of coating or kuboid kolumner simplex (a well-developed) wrapped by the thecal layer (composed of flat cells plated) ( ) = 30 µ

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Mature / ripe stage Visible enlargement of cortical alveoli and yolk granules. Oocyte size increases. Visible Migration of the nucleus towards the periph-ery / edge of the oocyte. Radiata zone layer visible light / bright. Folli-cle cells form a cube or a low cube surrounded / covered by a thin layer of thecal ( ) = 100 µ

Figure 3.  Description of histological structure of each oocyte development stage Brek and Lukas encountered in the female gonads, Harris Hematoxylin-Eosin

Figure 4. The proportion of each stage of spermatogenesis in Brek and Lukas fish in each period pre-domestication

The proportion of spermatogenesis in Brek and Lukas fish in the period pre-domestication.In the period pre-domestication 1 month and 2 months, the testes of fish Brek and Lukas are at initial stages of ripening, the small amount of spermatogonia, primary spermatocytes and secondary spermatocytes in relatively large quantities, but the number of spermatozoa little. While at the end of the period pre-domestication 4 and 8 months (Figure 4), has reached mature stage / RIPE stage, partly lobula lumina dominated by spermatozoa with large quantities. Spawning activity during the period pre-domestication, has carried out three times of induction of spawning, ie at the end of the period pre-domestication 2 months, 4 months and 8 months (Table 5). This activity is carried out to test the ability to do the breeding male and female post-pre-domestication in natural pond conditions ex situ.Based on the results of this activity is expected to note the readiness of the parent-parent post pre-domestication, as the basis for future assessments of early cultivation.

Table 5. The fish data is attempted to be cultivated with the help of induction ovaprim at the end of the period pre-domestication 1, 2, 4 and 8 months

No Date of spawning / fish species

Description

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3-06-2009End of

Pre-domestic

ation 2 months

Brek

1 head of female parent (400 grams): 3 heads of male parent (average of 250 grams), 6 hours after penginduksian, female parent died (IKG is already quite high, namely 19), histological observations showed female gonad preparations have reached the stage vitelogenesis towards mature (done in the pool experiment), did not work spawn

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7-07-2009End of

Pre-domestic

ation 4

Lukas

3 heads of mature female sex (± 60 grams): 5 males (± 50 grams), ± 10 hours after induction ovaprim have spawn partially, successful, 8 hours after the eggs have swelling, some egg-yolk egg yolk content of his other exit of eggs. All the female parent died 1 hour after Spawning. Histological observations of gonadal female parent has

BREK LUKAS

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monthsreached the stage vitelogenesis (done at the aquarium size 30 x 60 x 50 cm

324-10-2009

End of Pre-

domestication 8 months

Brek

Two mature females sex (± 225 grams): 5 males (± 100 grams), 11 hours after induction ovaprim have spawn successfully, all female or male parent either healthy post spawning (done in tub-sized 2.5 mx 1 m). The eggs results distributed and stocked in hatchery tanks and a few aquarium, successfully hatched and life

Lukas

4 females mature sex (± 75 grams): 4 males (± 50 grams), 12 hours after induction ovaprim have spawn successfully, all female or male parent either healthy post spawning (carried in tanks measuring 2.5 mx 1 m). The eggs results distributed and stocked in hatchery tanks and a few aquarium, successfully hatched and life

Fish Feeding Habits test. Plankton found in the swimming test during the study there are 54 genera (22 genera of zooplankton and phytoplankton 32 genera). Zooplankton consists of (6 genera) of the Class Rotifera, 8 genera of Class Crustaceae, 4 genera of the Ciliate Class and 3 from Class Sarcodina genera. While phytoplankton (2 genera) of Class Euglenophyceae, 8 genera of Class Cyanophyceae; 11 genera of the class Chlorophyceae and 11 genera of the class Bacillariophyceae (Table 6).

Feeding Habits of fiish test. Plankton found in the swimming test During the study there are 54 genera (22 genera of zooplankton and phytoplankton 32 genera). Zooplankton consists of (6 genera) of the Class Rotifera, 8 genera of Class Crustaceae, 4 genera of the Ciliate Class 3 from Class Sarcodina genera. While phytoplankton (2 genera) of Class Euglenophyceae, 8 genera of Class Cyanophyceae; 11 genera of the class Chlorophyceae and 11 genera of the class Bacillariophyceae (Table 6).

No Feed typeNumber of Index of Preponderance (%)

Brek ponds Lukas ponds1. Zooplankton 10,632 9,9862. Fitoplankton 56,893 62,8543. Undetermined material 0,518 0,0484. Worms - 7,6225. Animal cuts 0,03 0,0026. Pieces of plants 28,893 1,5287. Gastropoda - 15,0238. Detritus 1,268 1,264

Observations of fish digestive tract contents Brek and Lukas found 51 genera (nine genera of zooplankton and phytoplankton 42 genera). Zooplankton consists of 2 genera of Rotifera Class 3 from Class Ciliate genera, 2 genera of Class Sarcodina. Meanwhile, two genera of phytoplankton Class Euglenophyceae, 10 genera of the class Cyanophyceae, 17 genera of the class Chloro-phyceae and 13 genera of the class Bacillariophyceae. Research search hormonal profiles, activity spec gametogenesis and ecological Brek and Lukas are caught in the River Serayu aims to dig up the information beginning hormonal profile and the level of reproductive development of fish catch pre-domestication in aquaculture ponds, before the fish is ready for propagation .  Efforts pre-domestication fish from the environment in situ (river, sea etc.) into his new ex situ environment (eg aquaculture ponds) will always be followed by a series of processes of adap-tation to new conditions, including the effects of exposure to photoperiods in the new environ-ment. This work is done by Kujawa et al (1999) on wild fish in endangered status in Poland, which is domesticated through hatchery pond treatment model for stem mature gonad with artificial spawning.  In the wild, in waters such as rivers, lakes, seas, the activity of the reproductive physiology of fish that live in it is strongly influenced by local fotoperiodisitas (Zanuy et al. 1995). Zanuy fo-toperiode observe the influence of bright days are long and dark days of the hormonal profile of plasma 17β-estradiol (E2) and the process vitelogenesis ovary, fecundity, spawning time and egg quality of fish sea bass. Relative fecundity of the fish exposed to short daylight similar to the control

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group 257 000 230 000 eggs with eggs / kg female parent is ready mijah. But in the group of fish exposed to daylight length relative fecundity decreased up to half of the value of the control group fecundity (124,000 eggs).  Hormonal profile teleostei fish, both marine and fresh waters of the (non-farming) have been reported by several Frantzena et al (2004), Skjæraasen et al (2004), Shimizu (2003); Zanui et al (1995). All these authors report research on the relationship fotoperiode influence on hor-monal profile and the development of fish reproduction test.But the level of some hormone test fish were obtained from groups of fish caught in natural waters from various periods of the season, for

fish from the tropics is less information.  Data profile of the four types of hormone fluctuations in Brek and Lukas males and fe -males are shown Table 2 and Table 3, while the value of Gonad Maturity Index in Figure 1. Pre-view gametogenesis activity both oogenesis and spermatogenesis showed that an increase in these parameters until the end of the period pre-domestication 8 months. Although it is not fol-lowed by successful induction of spawning at the end of the period pre-domestication 1, 2, and 4 months, because only the Lukas parents who have spawn successfuly at the end of the period pre-domestication 2 months, but at the end of the period pre-domestication 8 months, Brek and Lukas parents both have spawn successfully (Table 5).  Reproduction in fish is influenced by environmental factors that control the endocrine glands that play a role mainly in the formation of reproductive hormones necessary for gonadal de-velopment, gametogenesis and the reproductive cycle (Fujaya, 2002).Shimizu (2003) explains that the process of reproduction, preparation of cooking fish sex until the spawning period is influenced by several environmental factors, photoperiods and temperatures. Further explained that in the dry season (summer) with a short-day light (≤ 13L), the strong decline of gonadal start the cessation of spawning period. But the two researchers Fujaya and Shimizu, examine only the fish in subtropical regions are of course very different from the fotoperiode tropois area.  Pre-domestication test Process on two fish in this study began in February 2009.  Until the end of February 2009 the last rain still fell in the research location, the village Kutosari Batu-raden District, Banyumas, although the frequency is not every day. Starting in March 2009 to Au-gust 2009 in the areas of Banyumas have started entering the dry season.Nevertheless pond wa-ter input experiment never lacked, given the location of the pool is very close to water sources orig-inating from Baturaden. As with other tropical fish, river fish course Brek and Lukas are naturally conditioned in the natural range fotoperiode 12L: 12D to 14L: 10D and even up to 16L: 8D.  17β-estradiol hormone profiles of the two groups at the end of the test fish pre-domestication 2 (P2b) and 4 months (P4b) decreased compared to initial levels in pre-domestication 1 month (Table 2 and Table 3). Profile hormone progesterone, FSH and Testosterone in fact the relative increase from the period of 1 month until the end pre-domestication period of 8 months (P8b). Pre-domesti-cation period of 2 months (P2b) began in March-May, pre-domestication 4 months (P4b) began in February to June, while pre-domestication 8 months starting in February - October. Period P2b and P4b are in the dry season, where the long bright days can be a maximum (≥ 14L).  A decline in levels of 17β-estradiol from the period of P1b, P2b, but in the P4b and P8b levels are actually increasing along with increased levels of the hormone progesterone ranging from period P2b, P4b until the end of the period P8b. This situation is an interesting thing because the proportion of oocytes vitelogenic stage Stage (v) at the end of the period of P4b and P8b actu-ally continued to increase (Figure 2). According to Biswas et al (2005) 17β-estradiol hormone pro-duced in the ovaries in part will go to the heart to stimulate the synthesis vitelogenin. Vitelogenin produced by the liver will be taken to the gonads via the bloodstream and internalized into the oocyte cytoplasm in the process vitelogenesis (Fujaya, 2002). Vitelogenesis process will result in increase of the volume of the oocyte as a process perubahanan oocyte developmental stages to mature stages of pre vitelogenesis (Arukwe and Goksoyr, 2003; Çakici and Üçüncü, 2007). In this case the pattern of hormonal regulation of both types of fish Brek and Lukas can not be explained, because in this study was not carried out measurements during the period pre-domestication vitel-ogenin levels. Therefore, research has yet to explain whether the profile of vitellogenin (fluctuation) follow the fluctuations in 17β-estradiol and progesterone.  Hormone levels of testosterone and FSH in both types of test fish and Lukas Brek in -crease from the period of P1b, P2b, P4b until P8b. In general, groups of fish from spawning Fa-milia Cyprinidae can do throughout the year as well Nilem (Osteochilus hasselti) and tawes (Pun-tius javanicus). Facilitation of testosterone and FSH in male fish and female gametogenesis is trig-

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gered toward gonadal maturation.  There are delays in the process of oogenesis both fish species tested in this study is to achieve a mature stage in the parent-parent female. Judging from Figure 2, proprosi oocytes that close to 40% - 50% of new-parent can be reached at the end of the female parent pre-domestica-tion period of 8 months (P8b). As is known, tropical fish from groups like Nilem Cyprinideae, tawes which is also one family with Brek and Lukas (materials research) can do more or less re-spawning after 2 to 3 months of previous spawning.This is what makes the question, why in the fish pre-do-mestication on research (especially Brek and Lukas who may have managed to spawn in the river, likely), it is not so and it took quite long (8-9 months) after the pre-domesticationkan in pond culti-vation of original place in the river Serayu. Possibilities to explain the occurrence of stress on the fish pre-domestication. In the first two months of acclimatization pre-domestication in the pool, the test fish did not want to consume food (pellets, leaf sente, and cassava leaves) are given. The process of adaptation occurs after entering the feed end of the second month that is willing to con-sume pellets, at the end of the third month, just started to consume additional food. Further note that the fish are experiencing stress will increase the secretion of glucocorticoid hormones from the adrenal glands, the secretion of glucocorticoids will inhibit the secretion of gonadotropins from the pituitary gland (Matsuwaki et al., 2006). Thus things may have happened in this study occurred be-cause of acclimatization the fish with his new environment. Many fish species in conservation programs toward domestication experience reproductive dys-function (Kouril et al., 2008). The case with the group that is difungsi familia Cyprinidae reproduc-tion is characterized by a lack of ability to reach the final oocyte maturation (Yaron, 1995; Mananos et al., 2009). After successfully completing vitellogenesis stage, the fish were unable to continue gamatogenesis phase and subsequent ovulation. Things like this is what may happen in Brek and Lukas fish in this study. Increased levels of the hormone progesterone, FSH and testosterone in the period up to 4 months pre-domestication (P4b) has not been able to support the attainment of maturity stages of oocytes from these parent-parent.  Therefore, an artificial environment such as conservation of fish ponds here are a few types of fish family Cyprinidae show reproductive endocrine dysfunction (Kroupova et al., 2005; Sudova et al., 2007), which is mostly at the level of final oocyte maturation (Yaron, 1995). This is caused by the secretion of the hormone LH that is not enough of the pituitary gland (Mananos et al., 2009), which is required for the activation of steroidogenesis and FOM (Kouril and Kodorek, 2007).  Judging from the histological observation of ovaries / female gonads and testes, then Brek and Lukas fish as other similar groups of Cyprinidae are grouped into multiple groups of spawners (multiple-batch group-asynchronous spawner) (Mananos et al., 2009), with characteris-tics met various or all stages of development oogenik in female gonads and testicular spermato-genic. IKG count results on the female parent of Brek and Lukas which can reach values 16% - 19% in the period ahead of final oocyte maturation mijah. In the observation in the pool, 2 female tails Brek who have achieved high IKG value ± 19% until the end pre-domestication 8 months yet to spawn, and even then died a few after being given induction treatment ovaprim several hours post-induction. Female parent who died of this, the histological observations of gonad both had reached the mature stage oocye with the proportion of 83.7%. The possibility that there is over-RIPE on her eggs, while it triggers stimulation from the environment to spawn yet or not soon reached. However, for other female parent (like the parent who has managed to spawn in late post-induction ovaprim pre-domestication 8 months) on October 25, 2009 (Table 5), on the one parent who sacrificed (not participate breeded) for purposes of observation, has a value of 16 IKG % with histological picture of the gonads had reached the ripe / mature oocytes as well, but with a lower proportion of 60.6%.  According to some researchers that deal with conservation of the fish from the Cyprinidae (Alok et al., 1997; Kouryl et al., 2008) induction to parent female parent using Canadian ovaprim preparation containing a GnRH + domperidone 0.5 cc / kg body weight, as we did in this study is very helpful to overcome the occurrence of reproductive dysfunction in the inability of the parent reached the final stage (final oocyte maturation), which then can successfully complete on spawning.  In the test fish food habits of Brek and Lukas have been done (Table 6), it can be ex -plained that there is little difference between pool maintenance Brek and Lukas. At the pool Brek pieces of plants and phytoplankton are as the main feed, zooplankton as food supplements, animal

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pieces and detritus is an additional feed. In Lukas phytoplankton pool is the main feed, zooplank-ton, worms and gastropods as a feed supplement, animal cuts, plant detritus and pieces as addi-tional food. However, the results of this test can be used to confirm whether the test fish had fully utilize the types of natural food in ponds, as a condition for the success of adaptive capacity with new conditions ex situ.  We still have some stock of Brek and Lukas parent couples who have been through a pe-riod of almost 9 months pre-domestication. On another occasion after spawning larvae seed parent of Brek and Lukas are still alive we still maintain today hopefully to be followed by subsequent studies until the age of first sexual maturity, while the parent-parent Brek and Lukas in the pool can be a super "predomesticated parent " . 

Conclussion

1.  The process of acclimatization to the pattern pre-domestication reached the stage of work, seen from the Index of preponderance (natural food preferences) in experimental ponds to reach the range of 37% -63% (phytoplankton); 20% -39% (pieces of plants), 8.75% -10 , 79% (zooplankton), 0.2% -14.9% (detritus, gastropods, worms and pieces of animals);

2. Brek fish hormone levels and Lukas: 17β-estradiol decreased from the period of P1b - P4b and increased at the end of the period P8b; progesterone, FSH and testosterone continue to increase the period of P1b - ending P8b;

3. Histology oogenesis, ovaries composed of 5 groups of oocyte developmental stages: chromatin nucleolar stage (CNS); perinucleolar stage (ps); cortical alveolar stage (cas); vitellogenic stage (vs) and mature / RIPE stage (ms), while spermatogenic testicular histology consists of 5 groups of stages: spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa;

4. At the end of the period of 8 months 2 tails pre-domestication parent Brek: 5 successful breeding male, 5 pairs of parent Lukas managed to spawn after the induction of sGnRH + domperidone 0.5 cc / kg body weight (ovaprim), ± 10-12 hours post-induction.

Acknowledgement

Our thanks goes to the National Research Council and the Ministry of Research and Technology, which has provided funds Incentives Research and Technology Research Association in 2008-2009 so that we can complete the research and reporting on time

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