Reproductive Synchronicity of Amblyceps Apangi in the Hill Stream of Arunachal Pradesh, India

NeBIO I www.nebio.in I December 2019 I 10(4): 209-219

RESEARCH ARTICLE Reproductive synchronicity of Amblyceps apangi in the hill stream of Arunachal Pradesh, India

Akash Kachari1*, Jyotirmoy Sonowal1, Manash Pratim Dutta1, Mahammed Moniruzzaman2 and Debangshu Narayan Das3 1*Freshwater Biology Research Laboratory, Department of Life Sciences, Dibrugarh University, Assam, India 2Fish Endocrinology Research Unit, Department of Zoology, University of Calcutta, West Bengal, India 3Fisheries and Aquatic Ecology Unit, Department of Zoology, Rajiv Gandhi University, Arunachal Pradesh, India *Email: [email protected]

ABSTRACT

The spawning periodicity of catfish Amblyceps apangi were determined from 502 individuals collected from the wild hill streams of Arunachal Pradesh, India. The macro anatomical index of gonad suggested A. apangi to spawn from May onwards till August with highest frequency observed in June (male) and July (female). Seasonal histological endeavor of gonads highlighted 5 developmental stages till the formation of mature spermatozoa and oogonial development occurred through 6 distinct stages. The sequentialcytostructural progression towards maturation that was observed using histological tool validated the findings of morpho-anatomical study. In addition, a non-invasive techniques (ELISA) using gonadal steroid (17 β-estradiol, 11- ketotestosterone and Testosterone) was also incorporated to predict the spawning periodicity of this fish. ELISA findings and their statistical enumeration with GSI% further approved that there exist a significant relationship between GSI %

development pattern of males with that of T (r = 0.87), 11- KT (r = 0.83) and of females GSI% with E2 (r=0.88). The study provides details about the reproductive periodicity of the hill stream catfish A. apangi in the wild, the findings is expected to be expedient to conservationist aimed at formulating conservation strategies.

KEYWORDS: Spawning, reproductive periodicity, ELISA, Testosterone, 11-ketotestosterone

Introduction merits not only in defining the exact reproductive seasonality in Gonadal maturity is a dynamic metabolic activity involving fish but is also instrumental in characterizing tissue types, organ sequential changes in the gonadal tissues, comprised of germ and organ systems, detect alterations in tissues, verification of cells and detailed characterization of those changes, helps sex and organs (Couch et al., 1974; Hinton and Couch, 1984; predicting the precise gonadal state of an organism (West, 1990). Blazer, 2002). Further, this technique has also proved its The reproductive timing of fish as evident from morpho- significance in categorizing the types of spawning (‘Isochronal’ or anatomical study could be justified by microscopic endeavor, ‘heterochronal’) in fish (Saeed et al., 2010). which allows proper discrimination and precise unambiguous grading of maturational status (West, 1990). Booth and Weyl According to Huggardet al. (1996), sexual maturity and gonad (2000) postulated that, reproductive strategies enumerated on the synchronicity shows a good correlation with blood steroid level. It basis of morpho-anatomy must be validated through histological has been reported that in teleost fishes, three most important endeavor if errors are to be minimized in predicting the exact steroids viz. 17 β-estradiol (E2), 11-ketotestosterone (11-KT) and maturity and reproductive seasonality of a fish.This technique Testosterone (T) produced in gonad perform all the critical steps

Received 9 November 2019 I Accepted 17 December 2019 I Published online 27 December 2019

Citation: Kachari, A., Sonowal, J., Dutta, M.P., Moniruzzaman, M and D.N. Das. 2019. Reproductive synchronicity of Amblyceps apangi in the Hill Stream of Arunachal Pradesh, India. NeBIO 10(4): 209-219.

Acknowledgements First author acknowledges University Grants Commission, New Delhi, India for providing financial assistance under the scheme Dr. DS Kothari Post Doctoral Fellowship.

NeBIO, An International Journal of Environment and Biodiversity Official publication of North East Centre for Environmental Education (NECEER), Imphal I ISSN 2278-2281 (Online), 0976-3597 (Print) I www.nebio.in

Kachari et al » Reproductive synchronicity of Amblyceps apangi in the Hill Stream of Arunachal Pradesh, India NeBIO 10(4): 209-219 of gametogenesis (Wallace and Browder, 1985; Miura et al., extracted using heparinized syringe from the caudal vein region of 1991; Agahama and Yamashita, 2008) and these are important the fish. Plasma was extracted using centrifuge (3000 x g for 10 markers of seasonal reproductive cycle. The rhythmic alteration in min) and stored at -20ºC until further assay. 17-β estradiol (E2) the gonadal steroids concurrently with that of developing gonads was measured using Enzyme Immunoassay kit, as per instruction have been investigated and found useful in understanding the manual (BiocheckInc, 837 Cowan Road, Burlingame, Ca 94010) mechanisms of gametogenesis and gonadal steroidogenesis and in house (Fish endocrinology unit, Calcutta University)

(Fostier, 1983; Goetz, 1983). In many teleost, the relationship prepared E2 standard in buffered matrix. Testosterone (T) was between seasonal changes of gonads and gonadal steroid level in measured using Enzyme Immunoassay kit (BiocheckInc, 837 plasma has been used as a tool for understanding the endocrine Cowan Road, Burlingame, Ca 94010), as per instruction manual regulation of reproduction as well as to determine the time of and in house (Fish endocrinology unit, Calcutta university) spawning (Pavlidis et al., 2000). In that context the present study prepared E2 standard in buffered matrix. 11-keto Testosterone is aimed at delineating the spawning periodicity of Amblyceps ELISA Kit is a competitive assay that can be used for apangi by means of histological endeavour, monitoring the quantification of 11-keto testosterone in plasma. The assay has a seasonal changes ingonadal steroid and further, its relationship range from 0.7-10 ng/ml and sensitivity (80% B/B0) of with the seasonal gonadal development cycle. approximately 1.3 pg/ml. This assay is based on the competition between 11-KT and an 11-KT-acetylcholinesterase (AChE) Materials and methods conjugate (11-KT tracer) for a limited number of 11-KT –specific Fishes were directly collected (using electrofisher and serum antiserum binding sites. The concentration of the 11-KT indigenousmethods for catching hill stream fishes) from different tracer is held constant while the concentration of 11-KT varies, wild hill streams of Arunachal Pradesh and brought to the wet the amount of 11-KT racer that is able to bind to the rabbit laboratory facility of the Fishery and Aquatic Ecology Unit, antiserum will be inversely proportional to the concentration of Department of Zoology, Rajiv Gandhi University, Arunachal 11-KT in the well. This rabbit antiserum-11-KT (either free or Pradesh, India for further study. tracer) complex binds to the mouse monoclonal anti-rabbit IgG antibody that has been previously attached to the well. The plate The gonadosomatic index was calculated as the percentage of is washed to remove any unbound reagents and then Ellman’s gonad weight to body weight (de Vlaminget al., 1982). Reagent (which contains the substrate to AChE) is added to the well. The product of this enzymatic reaction has a distinct yellow GSI = X 100 color, determined spectrophotometrically, is proportional to the where “GW” represents the weight of the gonads and “TW” amount of 11-KT tracer bound to the well, which is inversely is the total weight of the fish. proportional to the amount of free 11-KT present in the well during incubation. As far as the histological analysis is concerned, the preparation of slides of gonads and processing were done by adopting standard Results protocol (Lal, 2001; Abou-Seedoet al., 2003).A five point maturity Gonadosomatic Index scale common to most of the tropical spawner was adopted for For the estimation of GSI, both male and female specimen (n=502) the classification of the maturity stages of fish (Nikolsky, 1963; were sampled from January 2016 to December 2017.GSI showed Qasim, 1973; Owiti and Dadzie, 1989; Nunez and Duponchelle, an increasing trend from January onwards, attaining its peak in 2009), the stages were observed and photographed using Leica June (in male), July (in female) and thereafter showed a DM 500 B microscope.For Enzyme Linked Immunosorbant Assay decreasing trend. Maximum GSI (%) for male (Figure 1) was (ELISA)males and females fishes were captured from wild recorded in June (2.75 ± 0.40) and minimum in December (0.20 ± seasonally. From this wild caught stock, blood samples were 0.12).

Figure 1. a) Relationship between GSI (male) and Testosterone (T) in A. apangi. B) Relationship between GSI (male) and 11- ketotestosterone (11-KT) in A. apangi.

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Female (Figure 2) also followed a similar trend with maximum stage. The basophilic nature of cytoplasm lost its affinity and value recorded in July (21.07 ± 3.01) and minimum in December faintly stained therewith. A deeply stained somewhat spherical (0.39 ± 0.31).The matured phase (May – August) was body appeared in the cytoplasm just outside the nuclear characterized by increased GSI, where GSI value were membrane. It was the “yolk nucleus” or “Balbiani bodies”, significantly higher than the preparatory phase (January – April) appearance of this body marked the end of the primary growth which remained above 1% in male and 4% in female. phase and initiated the secondary growth phase i.e. Degenerative phase (September – December) was marked with vitellogenesis. GSI showing a declining trend and reaching its minimum during December in both the sexes. Cortical alveoli (S3) This stage was marked by the appearance of clear miniature vacuoles or vesicles (cortical alveoli) in the cytoplasm. These empty vascular structures were randomly distributed in the cytoplasm and were conspicuous when migrated towards the peripheral region (Figure 3c). The oocytes at this stage were less basophilic compared to S1 and S2 and stained pinkish blue. These oocytes were presumed to be in the early yolk vesicle stage. Yolk deposition caused the growth of oocytes in size ranged 250-400 µm, concurrently with the sizes and numbers of yolk vesicle until occupying the whole of the cytoplasm. The nucleolus which was distinguishable at the previous stage reduced in size and zonaradiata initiated its gradual development.

Vitellogenesis (S4) Figure 2. Relationship between GSI (female) and 17-β estradiol Oocyte at this stage is also known as late yolk vesicle stage. At

(E2) in A. apangi. this stage due to deposition of yolk further growth of oocyte took place along with increase in number of yolk vesicle. Zona radiata Histological changes in gonads was developing at the preceding stage became distinct at this Gonads passed through a series of developmental and stage and differentiated into zona externa and interna. Theca and maturational stages to enter a stage, when it was ovulated and granulosa cells were also seen around the zona externa. At this fertilized for producing young ones. The processes involving stage lately extensive deposition of yolk takes place with the several complicated cytological changes were identified based on rapid development of oocytes to reach at size range 440- 700 µm staining properties of the different cell components at different (Figure 3d). stages. Post -Vitellogenic stage (S5) Ovary The size of the oocytes attained maximum size of 750- 1000 µm at The maturing oocytes were characterized into five different stages this stage. The sizes of the constituent yolk granules of the based on cellular shapes and sizes and changes in microscopic peripheral region became larger by the fusion of one or more cellular components etc. viz. primary growth leading to early (S1) granules to and ultimately occupied the central position of the and late perinucleolus stage (S2), cortical alveoli (S3), vitellogenic cytoplasm. With reduced nucleoli numbers and sizes, the nucleus (S4) and post-vitellogenic stage i.e. oocytes with intact germinal lost its spherical shape with irregular nuclear membrane. The vesicle (S5). Besides, one more stages were also identified where nuclear membrane started dissolution and gradually the nucleus oocytes underwent nuclear membrane dissolution stage (S6). migrated at the animal pole. The migration of nucleus towards the animal pole clearly indicated final maturity of oocyte. Perinucleolus stage (S1 and S2) Histologically, ovary at spawning showed oocyte at S5 stage The early perinucleolar stage (S1) was characterized by having a (Figure 3e). large centrally placed nucleus, several deeply stained nucleoli and scanty cytoplasm which depicted basophilic nature. At this stage Ripe stage (S6) oocytes were small (<220 µm) in size, polygonal in shape (Figure The nuclear polarization (i.e. migration of nucleus towards the 3a). Multiple nucleolus of varying sizes were seen scattered in the animal pole) deposition and fusion of yolk globules and finally nucleus with some located around the peripheral region. In late nucleus breakdown were observed at this stage (i.e. S6) perinucleolar stage (S2) there was marked increase in the size of characterizing the final maturation and proximity of ovulation. the oocytes and polygonal shaped oocytes seems to be more Monthly distribution of percent frequency at different spherical in shape than S1 stage (Figure 3b). Associated with development stages of oocytes during the season has been oocyte growth, nucleus also increase in size, the scattered depicted in Figure 4. nucleolus at S1 stage were more synchronized and neatly arranged at the peripheral region of the nuclear membrane at this

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Figure 3. T.S of the ovary of A. apangi showing different developmental stages of gonad a Early Perinucleolus stage (S1); b Late Perinucleolus stage (S2); c Cortical alveoli (S3); d Early Vitellogenesis (S4);eVitellogenic stage (S5); f Ripe Stage (S6). N: Nucleus, NL: Nucleolus, YV: Yolk Vesicle, MN: Migrating nucleus, Vtg: Yolk globules.

Testes nucleus. Histological slides showed mitotically dividing cells in Spermatogonia (S1) large numbers (Figure 5a) in support of testicular growth. In Stage 1, spermatogonia were the largest cells in the male germinal tissue. The cells were spherical in shape having a large, Primary spermatocytes (S2) round and centrally placed nucleus being stained with basic dye The primary spermatocytes were produced through mitiotic but showing less affinity to cytoplasm. The nucleous were large, division of spermatogonia. These were smaller cells compared to deeply stained and were situated almost close to the centre of the spermatogonia and remained as small groups or nest, with darkly stained and relatively small sized nucleus compared to S1 cells

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(Figure 5b). Unevenly dispersed chromatin matters were more fine and condensed and stained purple with haematoxylin-eosin, however, nucleolus were hardly visible at this stage.

Secondary spermatocytes (S3) The secondary spermatocytes (S3) were much smaller strongly basophilic though the cells were morphologically similar to S2 stage. The chromatin depicted more composed, condensed and appeared as a homogenous body (Figure5c). Figure 4. Percentage domination of different oocytes stages during oogenesis of A. apangi.

Figure 5. T.S of the testes of A. apangi showing different developmental stages: aSpermatogonia (S1); b Primary Spermatocytes (S2); c Secondary Spermatocytes (S3); d Spermatids (S4); e Spermatozoa (S5); f Spent (S6).S: Spermatogonia, PS: Primary Spermatocytes, SS: Secondary Spermatocytes, SD: Spermatids, SZ: Spermatozoa, ST: Spent.

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Figure 6. Seasonal variation (n = 5 sample/month) of steroid level in male and female of A. apangi. a: Comparative seasonal variation in the steroid (T, 11-KT) level of male and b: Seasonal variation of 17- β estradiol level in female A. apangi.

Spermatids (S4) elevation and GSI development (Figure1b) was observed. GSI The cell at S3 stage did not last long and instantly divided enumeration of male showed its peak in June which tallied with meiotically to produce the spermatids (S4) cells, depicted further elevated 11- KT peak (4.31 ± 0.26 ng/ml) which was documented reduction in size. These were irregular in shape, with highly in the same month. From the above findings it can be assumed basophilic chromatin bodies (Figure5d) and somewhat elliptical that there exist a significant relationship between GSI and nucleus. steriods (T &11-KT) concentration in male of A. apangi. The findings were further justified statistically which further approved Spermatozoa (S5) that there exist a good relationship between GSI and T (r = 0.87), Apparently, the mature sperm cells were formed at this final GSI and 11- KT (r = 0.83). stage with the end of mitotic cell division. At this stage, reorganization of nucleus and cytoplasm took place. These cells 17-β estradiol concentration in female of A. Apangi almost though looked much special but much reduced in size, strongly showed a similar pattern of seasonal variation (Figure6b) as that stained with condensed nuclear materials and with the absence of male and their steroid distribution (T & 11-KT). Female E2 level of unstained areas visible at stage S4 (Figure5e). peaked in July i.e. at the onset of monsoon and during the

spawning period. E2 level after spawning period showed a sharp ELISA decline i.e. from September and ultimately dripping to its Figure6 represents seasonal variation in Testosterone, 11- minimum in November (0.60 ± 0.21ng/ml). Female plasma Ketotestosterone and 17-β estradiol concentration (ng/ml) estradiol tends to recover from this degenerative period of post inA.apangi for the entire experimental period. From Figure6 it has monsoon and gradually started increasing from February i.e. pre- been assumed that increase and decrease in the concentration of monsoon and continued its trend till monsoon (Figure 2b). Further T and 11 KT in the plasma of male followed as similar pattern. female showed a gradual increment in its GSI (%) from January Statistical analysis also confirmed the finding indicating high and ultimately attained its peak in July and gradually declined degree of correlation (r = 0.92) between this two steroid in their thereafter. A similar pattern of E2 development was noticed in elevation pattern. Male T and 11 KT showed a gradual increase female A.apangi where it reach its peak (5.66 ± 0.40 ng/ml) from January-April and progressively decreased after the during the monsoon period i.e. July and gradually reduced spawning phasei.e. September-November. Both the steroid thereafter, till the advent of the next preparatory period. showed marked increase in their concentration just prior to spawning, reached its peak during June, and showed a declining Discussion trend thereafter. T level reached its minimum during December Thorough knowledge of the developmental stages of gonads to be (0.97 ± 0.31 ng/ml) whereas minimum level of 11-KT was used as the marker in predicting the breeding/spawning period of encountered during January (1.11 ± 0.21ng/ml). Figure1(a) fish seems a reliable tool. Gonadal maturation in fishes is represents the relationship between GSI (%) and concentration of triggered by environmental stimuli (temperature, rainfall etc.) with testosterone (ng/ml) in male. The concentration of T attained its a regulatory effect on their reproductive timing, which inturn is peak during June (6.36 ± 0.50 ng/ml), this peak coincide with reflected on the changes of morphology and cytology of gonads maximum GSI level of male. Plotting a scattered diagram for 11- (Takushimaet al., 2008; Miranda et al., 2009). In female fish, Ketotestosterone and GSI of male, a similar pattern of steroid change of season is more pronounced where growth pattern of

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The features of Secondary spermatocytes (S3), Spermatids (S4) and Spermatozoa spermatogenesis and oogenesis of A. apangi apparently matched (S5) respectively. The S1 phase contained only spermatogonial the annual sexual cycle as shown by Gonadosomatic indices and cells only; S2 phase showed spermatogonia, primary the pattern of gonadal maturation seems to be similar as reported spermatocytes; S3 phase contained primary spermatocytes and in other teleost (Chakrabarti and Gupta, 1994; Chakrabarti and secondary spermatocytes, S4 phase was marked by formation of Banerjee, 2015). Oogenesis occurred sequentially with changes in spermatids population and in S5 phase spermatid dominated the oocytes such as differentiation from oogonia, primary and germ cell population. These findings were congruent with those secondary growth of oocytes, and development of yolk vesicle, reported in other catfishes by Sundararaj (1958), Sathyanesan vitellogenesis, maturation and finally culminating the cycle in (1959), Khanna and Pant (1966), Cavaco (2005). However, from the ovulation. These sequential structural changes have been months of December to February spermatogonia cells were the categorized in majority of the teleost into five, six or eight stages only germ cell present and indicated clearly as resting period. In (Sathyanesan, 1959; Dutt, 1964; Guraya et al., 1975; Malhotra, testes, the spermtagonial cells were present throughout the Jyotiand Gupta, 1978; Nagahama, 1983; West, 1990; Fishelsonet season but with remarkably lower population compared to al., 1996; Gulsoy, Aytekin and Yuce, 2006; Koc (Yon) et al., 2008). breeding phase (Khanna and Pant, 1966). On the backdrop of the Seasonal reproductive strategy of A. apangi revealed that the presence of spermatogonial cells throughout the season, Hann oocytes development occurred through six distinct stages (Figure (1927) termed these cells as “dormant mother cells”, as these 3). The oogonia passed through six different maturational stages cells acted as a reserve stock. It was also referred by many viz. early perinucleolar stage (S1), late perinucleolar stage (S2), authors (Jones, 1940; Suzuki, Agostinho, and Winemiller, 2000) as Cortical alveoli (S3), Vitellogenesis (S4), Post-vitellogenic stage “reserve germ cell”. In such a situation, it is believed that the fish (S5), Nucleus breakdown (S6) to become ripe oocytes ready to be might have the possibilities of undergoing spermeogenesis at any spawned. The cytostructural notifications at different stages such time though found to be sexually matured at the onset on as appearance and size of oocytes, follicular layer, cell wall, monsoon (May and June). In monsoon the testicles were filled in nuclear, nucleolus stage and staining competence of various with spermatozoa where Resink et al. (1987) and Gaber (2000) had cellular component elucidated all these aspects in detail. The similar observation in C. gariepinus and Bagrus spp. despite the cytostructural alteration clearly supported the view that A. apangi presence of spermatogonia throughout the season. Sundarajaj breeds with the onset of monsoon i.e. in between June to August and Vasal (1976) opined that temperature and photoperiod and the development of oocytes from oogonia to tertiary yolk regulated reproduction, as accordingly Swarup (1958) found that vesicles require about 4-5 months. During January, February the anatomical maturity of testes can be attained at any time of the oocytes were in chromatin nucleolus stage, the germinal vesicle year whereas functional maturation obtained only during the at this stage was round and large in size which gradually breeding period. decreased in size and became irregular in shape with the progression to maturity. Most of the oocytes in the ovary at this ELISA technique was incorporated to draw a relationship between period were dominated by S1 type oocytes, with the culmination morpho-anatomical cytostructural observations to those of S1 stage S2, S3 and S4 started appearing in April. Although biochemical parameters to delineate reproductive identity of this within the developing oocyte, occurrence of more than three particular fish. The procedure needs no sacrifice the specimens as stages was simultaneously noticed, vitellogenic oocytes (S4) with required in other conventional methods of characterization of hypertrophied follicular layers and yolky depository oocytes were reproductive strategies. The gonadal steroids T, 11-KT and E2 predominant during May-June. Corresponding with GSI value with showed a substantial effect on the gonadal development and its peak in July, ovaries were filled with S5 yolk oocytes, S6 reproductive behaviour of this endangered hill stream fish and oocytes with an indistinct nucleus as a result high rate of yolk expressed that their fluctuation in the plasma level, according to deposition undergoing nucleus breakdown could be seen in July, seasonal variations and influenced the gradual manipulations of August indicating the onset of spawning season. All these visible reproductive traits. This change in the visible or morpho- microscopic observation were congruent with a group anatomical reproductive feature in response to function of sex synchronous oocytes development that indicated isochronal steroids became instrumental in predicting the actual reproductive spawning pattern in endemic cat fish of Arunachal Pradesh, India. timing of fish species under study.

The testes of A. apangi having paired elongated lampbrush 17 β estradiol (E2) secreted from the female gonads and the inter- structure showed a remarkable uniqueness in term of its shape, renal tissues were responsible for the stimulation vitellogenesis. spermatic pattern and maturation both visually and histologically This hormone was more pronounced during the vitellogenic phase during development. The histological details of testes to reach the fish at its recrudescent phase. The findings on E2 of

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A. apangi represented in Figure 2 reflected the same clearly and spawning phase, i.e. starting from February and reached its peak that this event of vitellogenesis has been reported by many in May (Figure 1. a,b) and thereafter gradually dropped down. The workers (Campbell, Walsh and Idler, 1976; de-Vlaming et al., rhythmic fluctuation of steroid also corresponded with the male 1980; Smith and Haley, 1988) in most of the teleost. It was also GSI development and showed its peak value in May and declined observed that this hormone remained undetectable for some post this period. The observation of Ameret al. (2001) also period i.e. in late post ovulatory just prior to the recrudescent revealed that the elevated steroid levels increased the GSI till phase, and was in concert with Cornish (1998) for Oreochromis active spermiogenesis. Fostieret al.(1983) was also of the view mossambicus. In many fishes, after the appearance of the that T and 11 KT characteristically elevated during spermatogenic vitellogenic oocytes, E2 level also begins to increase and period and started declining after the spawning period till the remained in its peak till the tertiary vitellogenic oocytes stage but advent of the preparatory phase. According to Weltzien et al. subsequently lowered down during post ovulatory and atretic (2002) the highest GSI and 11 KT plasma level revealed at the 4 th phase (Matsuyama et al., 1991; Koushaet al., 2009). The stage of the male germ cell development but both the count went evaluation on this fish clearly demonstrated this seasonality in the down abruptly at the 5th stage. Koldras et al. (1990) tried to study circulation of Estradiol (E2) promoted the development and the sperm production and steroidogenesis in the testes of C. maintenance of the female sexual character as the same was carpio, their investigation also found congruent with the present documented where elevation in the E level promoted (Cornish findings which concluded that GSI in general had a good 2 1998) oocyte development. The finding seems quite convincing as correlation with most of the maturation indices including sperm in this fish developmental pattern of oocyte were in conformity production and area of the cysts. with the hypothesis. Statistical analysis also reveal that there exist a strong co-relation (r = 0.88) between GSI (%) and E2 Thus the study provided first-hand information on the reproductive concentration. With the increase in E2 level GSI also increase and condition and gonadal synchronicity of A. apangi. The estimated both attained its peak in July (Figure 2) and gradually lowered GSI with a single peak in monsoon clearly suggested an annual down or remained undetectable justifying the fact that E2 increase synchronous breeding pattern, spawning once in a year with a and ovarian development had a parallelistic rhythmic character short breeding period culminating at the end of monsoon. (Cornish, 1998). Histological analysis of the gonads revealed that ovarian and testicular growth and cyto-structural changes towards gonadal Gonads and adrenal cortex are the organs responsible for development followed similar pattern as in other catfishes. The androgen production in higher vertebrates. In male fishes one of progress of oogenesis in A. apangi took place through six (06) the sites of androgen synthesis and secretion is the testes and stages, whereas spermatogenesis occurred through five (05) this organ can synthesize testosterone and 11- ketotestosterone stages for achieving the level of maturation.These stages of (Vermeulen et al., 1994). According to Fostier et al. (1983) breeding cycle clearly validated the progressive cytological and testosterone affect the sexual behaviour (release of male structural changes in gonadal tissues of the fish confirming pheromone to attract opposite sex) of the fish and also this reproductive synchronicity for this less known wild fish of the steroid seems to have effects on some steps on spermatogenesis state. These were confirmed further with necessary justification i.e. in Spermatogonial multiplication and formation of using ELISA technique for gonadal steroids cycle. Further, the spermatocytes (Billard et al., 1982). On the other hand 11 KT investigation also proved that morpho-anatomical, microscopic drives spermatogenesis and stimulate the development of and immunological parameters have a positive relationship with secondary sexual characters in fish (Borg, 1994; Schulz et al., each other providing evidence towards predicting the reproductive 2010). Seasonal variation in the plasma level of T and 11 KT is timing of A. apangi in-situ. depicted Figure 1, revealed that there was gradual increase in these steroid from preparatory to matured and finally decreasing References during the degenerative phase. Such seasonal changes in these Abou-Seedo, F.D. and Al-Kanaan, K.A. 2003. Histology of ovarian steroid levels have been documented in many freshwater teleost development and maturation stages in the yellow fin species (Fostieret al., 1983; Dye et al., 1986; Liley et al., 1986; seabream, Acanthopagrus latus (Teleostei: Sparidae) reared Scott and Sumpter, 1989; Prat et al., 1990; Pavlidis et al., 2000). A in cages. Kuwait Journal of Science and Engineering 30: 121- high degree of correlation was also noticed (r = 0.92) in the 137. pattern of elevation of T and 11 KT under present investigation. Agahama, Y.N. and Yamashita, M. 2008. Regulation of oocyte According to Sisneros et al. (2004) and Butts et al. (2012) this type maturation in fish. Development, Growth and Differentiation of concomitant elevation in these steroids is a common pattern in 50:195-219. male fish. The elevation of steroid was probably the driving Amer, M., Miura, T., Miura, C. and Yamauchi, K. 2001. factors responsible for the morpho-anatomical changes and Involvement of sex steroid hormone in early stages of testicular development (i.e. immature testes to maturity). A spermatogenesis in Japanese huchen (Hucho perryi). Biology comparative analysis of GSI of the male and the pattern of of Reproduction 65: 1057-1066. circulation of T and 11 KT revealed that there exist a positive Bhatt, V.S. 1968. Studies on the biology of some freshwater correlation between GSI (%) to that of T (r = 0.87) and 11 KT (r = fishes. Part VII. Heteropneustes fossilis Bloch. Indian Journal 0.83). The concentration of T and 11 KT gradually increased in of Fisheries 15: 99 -115.

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