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Journal of Fisheries science and Technology No.3 - 2016 SEASONAL OVARY DEVELOPMENT AND EFFECTS OF VITAMIN E ON EGG PERFORMANCES IN RABBIT FISH (Siganus guttatus) Pham Quoc Hung1, Phan Van Ut1, Nguyen Van An2, Le Minh Thong3, Ngo Van Luong3 Received: 02/6/2016; Revised: 30/8/2016; Accepted: 26/9/2016 ABSTRACT In this study, we have characterized the seasonal reproduction profile and described changes histology of ovary in female rabbit fish Siganus guttatus. Besides, we investigated how vitamin E may effect broodfish maturation and ovary performances. Broodfish were fed with supplemental diet of vitamin E at doses of 500, 750 and 1000 mg Vitamin E/kg feed during reproductive season from February to June 2015. The results indicated that additional feeding with vitamin E during the conditioning period of broodstock produced no statistically significant effects on gonadal development, fecundities, and egg quality. The absolute fecundities varied from 565,585 to 665,723 egg per female depending upon the size of fish. Generally the bigger the fish, the higher the absolute fecundity. The relative fecundities ranged from 1215 to 1356 egg per g of female. Ripe egg diameter varied between 455 to 480 mm. The GSI at the ovary stage IV varied from 5.6 to 6.6%. The study forms a basis for understanding the reproductive biology of a tropical marine finfish with increasing industrial prospects and sustainable aquaculture of this species in Vietnam. Keywords: Rabbit fish, vitamin, Siganus guttatus, reproductive biology, egg quality I. INTRODUCTION Rabbit fish (Siganus guttatus) is a tropical marine finfish species presenting in coastal waters in Vietnam (Le and Le, 2006). Relative to other tropical species such as grouper (Epinephelus spp), cobia (Rachycentron canadum), mangrove red snapper (Lutjanus erythropterus), red drum (Scyaenops ocellatus) and Asian seabass (Lates calcarifer) with a long history of cultivation, the rabbit fish is not yet really commercial cultured because the fingerlings are collected from the wild (Kongkeo et al., 2010). Therefore, an attempt to study broodstock domestication and seed production in captivity has been conducted in Vietnam, but with limited success (Nguyen et al. 2003, Le and Le, 2006). One of the major causal reasons is the fact that broodstock management and spawning induction techniques are still poorly known for this species. For these reasons, Nha Trang University - Vietnam Kien Giang University - Vietnam 3 Department of Agriculture and Rural Development of Ca Mau 1 2 72 • NHA TRANG UNIVERSITY seeking methods to improve maturation, ovulation, fecundity, spawning performance and larval quality is a focal point for hatchery operators and successful aquaculture production. Broodfish diet with vitamin supplement may help induce metabolism and then improve maturation (Sandnes 1991). The aim of this study is therefore to understand and improve the maturation and spawning practices of this species, thereby minimizing the dependence on wild - caught seed stock and contribute to sustainable aquaculture. II. MATERIALS AND METHODS 1. Experimental design One year old brooders with a length between 28 - 30 cm and 450 - 600 g/fish were selected. These were held in fiberglass tanks in conditions that were similar to the natural environment, i.e: water temperature: 27 - 320C, Journal of Fisheries science and Technology pH: 7.8 - 8.4 and dissolved oxygen (DO): 4.5 6 mg/l. The brooders were daily fed at 2 - 3% of their body weight on commercial barramundi pellets with a proximate composition of protein: 43%, lipid: 7%, ash: 16%, fiber: 5% and moisture: 11%. The study was conducted between February and June 2015. This period is considered as the main reproductive season for rabbit fish in Vietnam. Three groups comprising 30 females in each (90 in total) were randomly distributed into tanks at different treatments as following: Treatment 1: Control Treatment 2: Suplement 500 mg Vitamin E/ kg feed (E500) Treatment 3: Suplement 750 mg Vitamin E/ kg feed (E750) Treatment 4: Suplement 1000 mg Vitamin E/kg feed (E1000) 2. Sampling reproductive parameters 5 females from each treatment were anaesthetized in freshwater to measure weight, length to the nearest 0.1 g and 0.1 cm, respectively and for measurement of gonad and the calculation of gonadosomatic index. To obtain gonad maturation status, anaesthetized fish were checked for maturity with a catheter. For the assessment of the fecundity, ovaries were removed from female cavity. Fragments of the ovary from the posterior, middle and anterior parts (0.5 - 1 g) and all eggs at stage III and IV (yolk formation) were counted. Absolute fecundity (AF) defined as the total of the eggs at stage III and IV in the ovary and relative fecundity (RF) was calculated as RF=100 x AF x W - 1 (where AF is the absolute fecundity and W is the total weight of the female fish). Egg diameter was determined as the average of 50 egg measurements under microscope equipped with a micrometer. Fish were considered as mature when they had Special issue No.3 - 2016 soft abdomen, swollen, protruding and reddish genital and swollen papilla and reddish anus. The maturation status was determined as the ratio (%) between mature fish in relation to the number of all fish examined in each treatment group. The gonadosomatic index (GSI) was calculated as 100 x gonad weight (g)/total body weight (g). 3. Gonadal histology Fragments of the gonad from the posterior, middle and anterior part of ovary and testis were fixed in Bouin’s solution. The samples were dehydrated through a series of increasing ethanol concentrations and embedded in paraffin. The tissues were cut at 6 mm thickness and stained with Harris’ hematoxylin and eosin. The histological sections were examined with a Zeiss Axioskop 2-Plus light microscope (Zeiss Inc., Vienna, Austria) and photographed through a Nikon Camera Head DS-5M and a Nikon Camera Control Unit DS-L1. Gonad development in female and male rabbit fish was classified according to standard procedure as described in Table 1. Maturation stages were determined using both the Sakun (Sakun, 1954) and Bagenal (Bagenal and Tesch, 1978) index. 4. Statistical analysis The statistical differences in reproductive variables including gonad maturation, egg and larval diameter, egg biochemical composition and reproductive parameters were assessed using one-way analysis of variance (ANOVA). Least significant difference and Duncan’s multiple range tests at 95 % confident level (Post Hoc Test) was used to compare mean values within individual exposure groups. All computations were performed with the software of Statistical Package for Social Sciences Version 18 (SPSS 18). Values are expressed as mean ± standard error (SE) or standard deviation (SD). NHA TRANG UNIVERSITY • 73 Journal of Fisheries science and Technology III. RESULTS AND DISCUSSION Special issue No.3 - 2016 advanced oocytes appeared more opaque and larger, covering a large area of total 1. Seasonal ovary histology All female did not indicate ovarian stage I oocytes that are typical for immature teleosts as in the first event in their life cycle. During sampling in January and February, all female were classified as immature ovaries (stage II) with small proliferating oogonia, previtellogenic oocytes (primary and secondary oocytes). At the next sampling period in March, about ovarian volume. As oocyte development advanced into the peak season (April-September), stage III-VI (that include germinal vesicle oocytes, ovulated, post-ovulatory follicular cells and lipid droplet) became evenly distributed in the female oocyte. From October, stage II oocytes reappeared and were the only oocyte group observed in November and December. The females had residual eggs left 20% of the females had commenced cortical in their ovaries immediately after spawning alveoli formation and yolk sequestration, and these ovaries usually contained only making these oocytes semitransparent (stage III). previtellogenic oocytes during late sampling in As vitellogenesis proceeded, the most November and December. Table 1. Seasonal ovary development Month Ovary stage January II February II March II + III April III + IV May III + IV June III + IV + V July IV + V August II + VI September II + III October III + IV + V November IV + V December II The ovary histology indicated that rabbit oocytes entered the rapid growth phase fish have a multiple spawning pattern and long in April-June and July-September. The histology spawning period. They are probably coinciding with also showed it is in accordance with the recruitment of oocytes into yolk accumulation, multiple but increased significantly after the maturing developmental strategy 74 • NHA TRANG UNIVERSITY group asynchronous gamete Journal of Fisheries science and Technology No.3 - 2016 Stage II Ovary containing previtellogenic oocytes including early and late perinucleoli oocytes Stage III Ovary with vitellogenic oocytes including primary, secondary, and tertiary yolk characterized by the yolk globules and peripheral yolk vesicles. The nucleus is still in a central position. Stage IV Germinal vessicles (GV) starts to migrate from its central position to the periphery. GV completes its migration towards the periphery (animal pole) and a large lipid droplet Figure 1. Microscopic description gonadal development. Gonads are divided into the following six developmental stages based on the most advanced stage of gametes in the gonads 2. Effects of vitamin E on female gonadal performances The results of inspection of brooders for final oocyte maturation and ovulation (FOMO) every 15th day indicated that under captive condition, fish could reach the FOMO, but not frequently and at a low rate. The fish basically reached maturity in late spring from April to May. No statistically significant differences (P>0.05) in FOMO were observed among the treatment groups (Table 2). NHA TRANG UNIVERSITY • 75 Journal of Fisheries science and Technology No.3 - 2016 Table 2. Results of inspection for oocyte maturation and ovulation in the female brood fish during the experiment period Tretament groups (mg vitamin E/kg of feed) Sampling date E500 E750 E1000 Control 15/03/2015 (-) (-) (-) (-) 01/04/2015 (-) (+) (-) (-) 15/04/2015 (+) (-) (+) (+) 30/04/2015 (-) (+) (+) (++) 15/05/2015 (+) (++) (++) (-) 30/05/2015 (++) (+) (-) (+) (-): Indicate immature as the ovary is at stage I or II (+): Indicate mature as the ovary is at stage III or IV (++): Indicate ovulation as the ovary at stage V Our study indicated that vitamin E did not influence oocyte maturation, ovulation and or reproductive performances in rabbit fish. Maturation in teleost species includes the breakdown of the germinal vesicle (GVBD), yolk clarification, and an increase of oocyte volume. Ovulation is the release of a mature oocyte from its follicle into the ovarian cavity. The effects of vitamin E on fish reproduction, especially via oral administration, is not well studied, and may vary depending on species, population, and life history strategies. The authors observed that the method of vitamin E supplemented with diets for rabbit fish brooders has not been reliable since feeding method may not be equal and diestability among individual. Hence further study on how to feed the brooders with vitamin should be conducted. Table 3. Effects of vitamin E on egg performances Reproductive parameters Average mature rate (%) Absolute fecundity (egg/female) Relative fecundity (egg/ g of female) Tretament groups (mg vitamin E/kg of feed) E500 E750 E1000 Control 26 ± 14 22 ± 12 24 ± 13 46 ± 18 570,575 ± 46,127 565,585 ± 56,127 610,552 ± 96,210 665,723 ± 88,623 1,356 ± 185 1,258 ± 165 1,215 ± 147 1.266 ± 165 GSI (%) 5.6 ± 1.5 6.5 ± 1.8 5.8 ± 1.7 6.6 ± 1.3 Ripe egg diameter (mm) 455 ± 56 465 ± 76 480 ± 65 458 ± 46 No significant differences of absolute and relative fecundities, GSI, matuaration rate and egg diameter among treatment groups were observed (P>0.05). The absolute fecundities varied from 565,585 to 665,723 eggs per female depending upon the size of fish. Generally the bigger the fish, the higher the absolute fecundity. The relative fecundities 76 • NHA TRANG UNIVERSITY ranged from 1215 to 1356 egg per g of female. Ripe egg diameter varied between 455 to 480 mm. The GSI at the ovary stage IV varied from 5.6 to 6.6 % (Table 3). The reproductive parameters mentioned above may be important as predictive and reliable tools of reproductive status that are integral aspects for broodstock management. Journal of Fisheries science and Technology These parameters will also be important for an accurate evaluation of the effects of different treatments on sexual maturation and spawning in fish farming. Herein, we report the effects of the vitamine E on spawning profile and gonadal maturation and development in female rabbit fish. For many years in the field of aquaculture scientists have attempted to develop techniques to induce reproduction in fish using hormonal stimulation (De Silva et al., 2008; Nguyen et al. 2013). In this study, we showed that vitamine E exposure of female rabbit fish did not show any differences in No.3 - 2016 enhancing reproductive performance among the treatment groups. IV. CONCLUSIONS Rabbit fish is a multiple spawer with a main spawning season from April to July. In the ovary it is obvious with the multiple group asynchronous gamete developmental strategy. In this study supplement of vitamin E in the daily diet for brooders during conditioning had no effect on gonadal maturation and ovary performances in rabbit fish. REFERENCES 1. Bagenal, T. B. & Tesch, F. W., 1978. Methods for assessment of fish production in fresh waters. In Age and growth (Bagenal, T., ed.). Oxford: Blackwell Scientific Publication: 101-136. 2. De Silva, S.S., Nguyen, T.T.T., Ingram, B., 2008. Fish Reproduction in Relation to Aquaculture. In: Fish Reproduction: Cytology, Biology and Ecology (M.J. Rocha, A. Arukwe & B.G. Kapoor, eds.). Science Publisher, Oxford, IBH Publishing Co.: 535-576. 3. Kongkeo H., Wayne C., Murdjani M., Bunliptanon P., Chien T., 2010. Current practices of marine finfish cage culture in China , Indonesia , Thailand and Vietnam. Aquac Asia Mag: 32–40. 4. Le, V.D. and Le, D.N., 2006. Reproductive biology of golden rabit fish (Siganus guttatus Bloch, 1787) in Thua Thien Hue province, Vietnam. Journal of Agriculture and Rural development No. 1: 49-61 (In Vietnamese with Abstract in English). 5. Nguyen, P. T, Bui, T M., Nguyen, T A., De Silva, S., 2013. Developments in hatchery technology for striped catfish (Pangasianodon hypophthalmus). In: Advances in aquaculture hatchery technology (G Allan and G Burnell, Eds.): 498-518. 6. Nguyen, T. N., Luc, M. D. & Nguyen, D. T., 2003. Study on seed production of Sand bass Psammoperca waigiensis (Cuvier & Valenciennes, 1828). In A research report under contract between the University of Fisheries and SUMA, Ministry of Fisheries, Vietnam 2003 (in Vietnamese). 7. Sakun, O. F., 1954. Analysis of gonadal function in male and female Vimba vimba L. with special reference to the nature of spawning. Dokl Akad Nauk SSSR 98: 505-507. 8. Sandnes K, 1991. Vitamin C in fish nutrition - a review. 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