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Histological investigation on gonad maturation of cultured short fin eel, Anguilla bicolor (mcclelland, 1844) in captivity.

Anguilla bicolor or commonly known as a shortfin eel is a subspecies of eel in the genus of Anguilla and widely distributed in South East Asia. Present study reveals the first report of the early gonad development of cultured A. bicolor in captivity via histological analysis. The total length and body weight of A. bicolor were 511 to 614 mm and 209 to 420 g respectively. The result showed all specimens were female with no sign of male or intersexual stage. Through histological analysis, identified female were classified in 1) immature (n=3) by the presence of oogonia (O) and primary germ cell (PG) and also 2) developing stages (n=11) by the presence of previtellogemic-stages oocyte (PVO), oogonia (O) and cortical alveolar oocyte (CAO). Meanwhile, the fin difference index (FDI) and eye index (EI %) ranged from 0.55 to 3.89 % and 1.64 to 4.56% respectively. This study concludes smaller size (511 to 614 mm) of cultured A. bicolor is immature, while gonad maturation was already in developing stage for larger size (565-614 mm), hence these range of A. bicolor are still unable to be used as broodstock.


Introduction
Anguillid eel species are generally distributed in tropical and temperate seas. To date, 15 species of Anguilla have been reported and seven subspecies alone can be found in Asia including Malaysia and Indonesia [1]. Anguilla bicolor or known as shortfin eel is among the most abundant and common species can be found. This species have an excellent organoleptic qualities and often attain high demand in many parts of the world. In Japan, A. bicolor is more preferred for consumption as "unagi kabayaki", a renowned cuisine of eel rather than other species of Anguilla after A. japonica, A. Anguilla and A. rostrate [2]. It is actually boned and filleted fish, grilled with a sweet Japanese kabayaki sauce, whereby only eel suits to produce this delicacy. Because of the continuous high demand of ungai kabayaki, there is many kabayaki processing factories has been expanded in South East Asia. However, the supply of A. bicolor are still highly depending on wild stock, since its artificial breeding techniques is not yet firmly established anywhere. The collection of wild A. bicolor has been banned since 2009 right after they are listed under Appendix II by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) in 2009. Hence, eel has now becoming new emergence of aquaculture species to suppress the exploitation of wild stock [1].
In aquaculture, study of gonad stages of maturation has become increasingly important in fish production, notably in induced spawning purposes. Hence, this study aims to investigate the sex and gonad maturation of A. bicolor in captivity in order to serve as primary data for its seed production in the future. There are plenty of studies have been carried out to understand gonad development of eels done including those by [3,4,5,6]. However, the findings on the development pattern of gonad remain a matter of debate among the researches around the world. Contradict to a study done by [7], sex differentiation of eel was related with size of eel (total length) rather than age. Findings by [4] Colombo and Grandi (1996) had revealed body size of A. anguilla are relatively smaller than 20 cm was actually undifferentiated, and those beyond 20 cm was female with no clear sign of intersexual stage and transformation into male stage. However, there is still a lack of study on gonad development, sex determination and relation with body size on tropical eel. Thus, the present study was aimed to investigate on gonad development and sex determination of A. bicolor through histological study and its relation with body size and other morphological characteristics. Fish reproduction information is important as primary guideline not only for aquaculture but to fisheries management as well.

Specimens Collection
A total of 14 specimens of cultured A. bicolor were collected alive from eel farming and processing factory, Pt. Iroha Sidat at Bali, Indonesia. All the specimens were identified and categorized in yellow stage where the pectoral fins of specimens was white or grey, green or grey fin and yellow or white belly. Prior to measurement and analysis, the specimens were transferred into laboratory and anesthetized with cold water method.

Gonad Maturation and Fecundity
Gonad maturation and fecundity were described according to method of [9], whereby it include measurement of gonad length (GL) in both left and right side. After measurement, weight of gonad (GW) was recorded. The Gonad Somatic Index (GSI) was calculated as follows: Gonad Somatic Index = (gonad weight/body weight) x 100

Morphological Study
The eel gonads were initially fixed in Bouin's solution for 24 hours and later dehydrated in series of ethanol. After that, sample was embedded in paraffin, sectioned to 7 µm thickness and then stained with Haematoxylin and Eosin. Gonad samples were mounted in glass slide and observed under light microscope (Nikon, Eclipse E600, Japan). Gonad maturity of eel was classified based on description developed by [10].

Morphological, Fin Index, Eye Index and Gonado Somatic Index
The FDI % ranged from 0.55 to 3.89%, with mean ± SD of 2.40±0.88% (Table 1). [1] found that FDI% A. bicolor bicolor in Peninsular Malaysia within the range of -1.93 % to 2.63 %, mean±SD of 1.06±1.20%. The value was in the range compared to previous study (range; -3 -3.9% in A. bicolor bicolor by [11]. Therefore, those specimens could be easily identified as the A. bicolor. Meanwhile, IE% was ranged from 1.64 to 4.56%, with mean±SD of 0.89 ± 0.57% (Table 1). According to [12], eye index or known as ocular index (OI) is very powerful indicator of transformation degree in gonadal development of eel. [13] revealed eye index of A. bicolor in range of 5.72 to 6.02% were high as it is approaching sexual maturity. Fully matured A. bicolor possess of eye index in range of 12.31% above and ready to leave freshwaters for spawning. The alteration of external morphology of the silver phase eel has been suggested as part of the preparation for the adaptation to the marine setting before their spawning migration occur [14]. The TL and BW of A. bicolor in the present study was range from 511 to 614 mm and 209 to 420 g respectively. Other morphological characteristics were provided in Table 2. Mean value of Gonado Somatic Index (GSI, %) in the present study was 0.89±0.57. GSI values of female A. japonica collected in the East China Sea ranged from 1.3 to 3.5 [15] meanwhile [16] reported GSI value of females A. japonica collected from Mikawa Bay and Amakusa Islands ranged from 1.0 to 4.3. Both studies showed that female A. japonica were in developing and maturation stages. Compare to the present study, these GSI values were relatively higher. [17] claimed that GSI value of female A. bicolor bicolor that collected from the Waters of Segara Anakan ranged from 0-3% and believed that the immature female and they still require longer time to develop as sexually mature adults before spawn in the ocean. Therefore it could be suggested that, GSI value of immature and developing stage of female A. bicolor ranged from 0-3%.

Histological Analysis-Gonad Maturation
Histological observation revealed that the samples in the present study were ovary because of the presence of oocytes. Based on gonadal classification by [10], the present female specimens were classified in immature and developing stages. The TL of immature specimens was 511 to 565 mm. Based on histological observation, the immature specimens were defined as presents of oogonia (O) and primary germ cell (PG) (Figure 1a . This stage also identified as oogonia and chromatin nucleolar stage where the ovary was composed of bunches of oogonia, oocytes which were believed to further development of ovarian sexual cells. Oogonia (marked as arrow in Figure 1a,b) are the smallest germ cells with no noticeable boundries, whereby the cells found either solitary or in bunches of lamellae adjacent to the germinal epithelium and was used to describe the gonadal development. [18] found that gonads of A. australis in the range of 75 -100 mm in TL were consisting of two to six primordial germ cells. Meanwhile in 100 -200 mm A. australis, gonads shift into a lamellar shape containing 5 -50 primordial germ cells which will again increase to bunches. As eels larger in size (75 -150 cm) the gonads tend to expand to lamellar shape and the density of primordial germ cells will eventually rise. In 150 -200 mm eels several primordial germ cells will increase to bunches of primordial germ cells and later lamellae also will increases in size.
Meanwhile the developing stage was well-defined by ovaries that possessed previtellogemic-stages oocyte (PVO), with oogonia (O) and cortical alveolar oocyte (CAO). The TL of developing specimens were normally between 565 to 614 mm. [10] found that A. bicolor in developing stages was 421 to 885 mm. At this time, the ovaries were enlarging because of the deposition of trophic substance such as yolk and fat. Early stage of ovaries were characterized by lamellae comprising double rows of previtellogenic oocytes at the perinucleolus stage, running from the vascular to the germinal region by a thin mesogonadium [4]. Figure 1e showed the oocytes in the previtellogenic stage, which comprising a large central round nucleus (or germinal vesicle), multiple nucleoli, and ample cortical alveoli that completely filled the cytoplasm. As the size of gonad upsurge, the cortical alveoli oocytes (CAO) are

Conclusion
In conclusion, cultured A. bicolor in the captivity with total length and body weight at 511 to 615 mm and body weight 209 -420 g respectively were female (immature and developing stages). There are no sign of intersex and male stage was observed. In the next study, variation of size is required to reveal the process of gonad development, sex differentiation and its relation with body size should be carried out.