Ovarian Maturation in the Macrobrachium nipponense by Serotonin Injection

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1 International Research Journal of Applied and Basic Sciences 2013 Available online at ISSN X / Vol, 4 (5): Science Explorer Publications Ovarian Maturation in the Macrobrachium nipponense by Serotonin Injection Mohammad Navid Forsatkar 1, Mohammad Ali Nematollahi 1,*, Maryam Hedayati rad 1 1. University of Tehran, Faculty of Natural Resources, Department of Fisheries, PoBox: , Karaj, Iran. * Corresponding Author malahi@ut.ac.ir ABSTRACT: This study was conducted to investigate the effect of serotonin (5-HT) injection as a neurotransmitter on ovarian maturation in female Macrobrachium nipponense. The neurotransmitter was dissolved in saline and injected at concentrations of 1, 4, 8 and 12 µg g -1 body weight (BW) in 1st, 4th and 8th day of experiment. Control group was injected only with saline. At the injection of 1 µg g -1 BW, 68% of prawns reached to stage IV of development, but this rate decreased in other groups of 5-HT treatment. No female in control group reached to this stage. In addition, ovarian index was significantly highest (6.19±1.16%) at 1 µg g -1 BW (p<0.05). The results suggest that 5-HT able to induce gonad maturation in M. nipponense. Keywords: Swamp shrimp, gonad development, 5-HT injection, ovarian index. INTRODUCTION The genus Macrobrachium includes several important species that exist in the brackish and freshwater habitats. Freshwater swamp shrimp, Macrobrachium nipponense is a small prawn with maximum length of 86 mm for male and 75 mm for female. It is originated from North China to Annam, Japan and Taiwan and recently found in Caspian lake basin in north of Iran (De Grave and Ghane, 2006). The females sexually mature at 4-5 months old and mating occur between newly molted females and hard shelled males (New et al., 2010). Reproductive systems of crustacean have been considered for many years. Evidence showed that reproduction and maturation in shrimps are under the hormonal control and these hormones regulate reproduction and spawning (Nagaraju, 2010). Several methods have been used to induce ovarian maturation in crustaceans. They based on inhibiting the release of gonad-inhibiting hormone and/or enhancing the release of gonadstimulating hormone (Fingerman, 1997). These Two antagonistic hormones were involved to regulate development of the ovary in crustaceans. The most common method is eyestalk ablation but this is not the most effective style because egg quality will decrease and spawner may be dies (Wongprasert et al., 2006; Varalakshmi and Reddy, 2010). The X-organ is present in this part of the body and contains sexual inhibitor hormones (Chen et al., 2003). It is known that biogenic amines especially serotonin (5-HT) regulates synthesis and releasing the reproductive regulator hormones, therefore it can affect on ovarian maturation and ovulation. Serotonin is derived from amino acid tryptophan in certain enzyme pathways distributed at high levels in optic ganglion, cerebral ganglion, circumoesophageal connectives, stomatogastric ganglion, and thoracic ganglia (Tinikul et al., 2008). Studies on fish and crustaceans showed that serotonin and selective serotonin reuptake inhibitors (SSRIs, such as fluoxetine) can affect on food intake (Ruibal et al., 2002), aggressive behavior (Clotfelter et al., 2007; Tierney et al., 2004), social relationship (Lepage et al., 2005; Peeke et al., 2000) and reproduction (Mennigen et al., 2008; 2010). Also, there are a number of studies carried out in some important shrimp species indicating the role of exogenous serotonin on reproduction performance and ovarian maturation (Alfaro et al., 2004; Meeratana et al., 2006; Santhoshi et al., 2009). Thus, any treatment with serotonin or its agonists which can increase serotonergic pathway are considered to stimulate ovarian maturation. This study was therefore aimed to investigate the effect of 5-HT on ovarian maturation in M. nipponense.

2 MATERIALS AND METHODS Animals Experiment was done in a private center for ornamental fish culture in Karaj, Iran. Number of 100 females (3-5 g body weight) from the same location was selected. They stocked in four 120 L glass aquariums. Prawns were fed twice daily with commercial pellets and frozen blood worms for two weeks acclimation. Temperature was C and water change about 50% every 3 days. To decrease aggression among animals, each tank equipped with 15 small clay pots. Procedure Prawns were randomly divided into five groups (n=10, 2 replicates). At four treatments they received 1 (5- HT 1), 4 (5-HT 4), 8 (5-HT 8) and 12 (5-HT 12) µg g -1 body weight serotonin creatinine sulfate (Sigma; obtained from Taleghani hospital, Tehran) in days 1st, 4th and 8th. Required amount of serotonin for each group weighted and dissolved in saline. Injections of 0.05 ml final volume for each animal were performed by micro-syringe at the base of pleopods of third abdominal segment. Control group was treated the same as the experimental groups but received injections of saline. At the end of the experiment on day 15, ovarian maturation of females classified into five stages based on the size and color of the ovary as reported by Meeratana et al. (2006). Stages 0 and I were spawned and spent phases when the ovaries appeared white. Stage II (proliferative) a small yellow mass of ovary could be observed dorsally in the carapace. Stage III (premature) the ovaries became orange. Stage IV. The reddish orange ovary extended from behind the eye posterior to the first abdominal segment. Finally animals sacrificed and average ovarian index (OI= ovarian weight / body weight 100) of the prawns in each group were determined. Statistical analysis Statistical significance differences were tested by one-way ANOVA followed by Duncan s multiple range test (p<0.05). Pearson correlation coefficients were used to assess relationships between 5-HT injection doses and stages of development. RESULTS Animals were tested for gonad development on day fifteen after first injection. According to Fig. 1, 70% of control group developed to stage II and 6% to stage III, while the rest remained at stage I. The ovaries of 5-HT 1 mostly reached to stage IV (68%) while 51%, 42% and 12% of 5-HT 4, 5-HT 8 and 5-HT 12 reached to stage IV, respectively. Most of 5-HT 12 prawns developed to stage III (56%) and stage II (20%). All of the developmental stages were positively correlated to 5-HT doses (Table 1). This correlation was significant at p<0.01 for Stages I and IV and at p<0.05 for stage II. Injection of serotonin causes maturation in M. nipponense (Fig. 2). At 1 g g 1 BW, 5-HT induced a significant increase in the ovarian index (6.19±1.16%) as compared to control group (1.44±0.51%), with significant different at p<0.05. Smaller increase of OI was observed in other 5-HT groups but it was generally lower when the dose of 5-HT was higher. DISCUSSION Several studies demonstrated that neurotransmitters especially serotonin can induce gonadal maturation in Crustaceans. Some of these examples are ovarian maturation and spawning in the black tiger shrimp Penaeus monodon (Wongprasert et al., 2006) and white shrimp, Penaeus vannamei (Vaca and Alfaro 2000), ovarian maturation and hemolymph vitellogenin in the Indian white shrimp, Fenneropenaeus indicus (Santhoshi et al., 2009) and in giant freshwater prawn broodstock, Macrobrachium rosenbergii (Meerantana et al., 2006; Tinikul et al., 2008). Furthermore, Alfaro et al. (2004) induced ovarian maturation and spawning in Liptopenaeus stylirostris and Liptopenaeus vannamei by combined treatment of 5-HT and Dopamine antagonist, spiperone. The results of the present study showed that injection of 5-HT can induce ovarian maturation and increasing ovarian index in female M. nipponense. The highest ovarian index (p<0.05) and maximum number of shrimp that matured to stage IV was observed at the dose of 1 g g -1 BW. This effective dose of 5-HT injected into M. nipponense was the same as M. rosenbergii results (Meerantana et al., 2006). The control role of gonad inhibitor hormone (GIH, released from the sinus gland in the eyestalk optic lobes) over the ovaries and

3 hepatopancreas is very intense (Vaca and Alfaro 2000), thus injection of exogenous serotonin cause prevention the release of this hormone and then ovaries going to develop. Inhibition of GIH release is not the only process for gonad developing but it is essential. In addition, serotonin is involved to stimulate the release of the molt inhibiting hormone, the hyperglycemic hormone, the red pigment dispersing hormone and the neurodepressing hormone (Wongprasert et al., 2006) in crustaceans. Based on the result, 5-HT was positively correlated with ovarian maturation in all groups (Table 1), but different number of prawns reached to different development stages. The number of females which developed to stage IV decreased with increase in 5-HT dose from 1 to 12 g g -1 body weight. Results showed that in 15 days experiment the ovaries of 5-HT 1 group mostly developed to stage IV (68%) while no prawn in the control group reached to this stage. This is because of 5-HT injection shorten the period of ovarian maturation (Meerantana et al., 2006). Evident showed that in shrimp, 5-HT act indirectly on the ovaries and it can be said that higher amount of 5-HT affect on the endocrine system in crustaceans. This effect leads to decrease both in final gonad maturation and ovarian index. As a result of this situation, serotonin is involved in the ovarian maturation of M. nipponense. There are different ways to induce maturation in shrimp species but it seems that injection of serotonin is a suitable method which can help the shrimp culturists to achieve mature broodstock in a shorter time. Also, induce of maturation by eyestalk ablation increases mortality and loss of egg quality, therefore 5-HT injection can be an efficient practical alternative for gonad maturation in the prawns such as M. nipponense. ACKNOWLEDGEMENTS The authors appreciate the support of Mr. Ashrafi s ornamental fish farm, from Karaj, Iran. Table 1. Pearson correlation coefficient among 5-HT injection doses and stages of development. Stage I Stage II Stage III Stage IV.964 *.999 ** * 5-HT dose (g g 1 BW) (.036) (.001) (.122) (.023) (p values in parentheses, n= 20; *p 0.05, **p 0.01).

4 Figure 1. Percentage of ovarian stages that the prawns attained 15 days after the first injection. Stage I- spent, stage IIproliferative, stage III- premature, stage IV- mature. Figure 2. Ovarian index (OI) of the Macrobrachium nipponense, at day 15th after multiple injections of 5-HT at 1, 4, 8, 12 g g -1 BW on day 1st, 4th and 8th. Bars with different letter superscripts are significantly different (p<0.05). REFERENCES Alfaro J, Zuniga G, Komen J Induction of ovarian maturation and spawning by combined treatment of serotonin and a dopamine antagonist, spiperone in Litopenaeus stylirostris and Litopenaeus vannamei. Aquaculture, 236: Chen YN, Fan HF, Hsieh SL, Kuo CM Physiological involvement of dopamine in ovarian development of the freshwater giant prawn, Macrobrachium rosenbergii. Aquaculture, 228: Clotfelter ED, O'Hare EP, McNitt MM, Carpenter RE, Summers CH Serotonin decreases aggression via 5-HT1A receptors in the fighting fish Betta splendens. Pharmacology, Biochemistry and Behavior, 87: De Grave S, Ghane A The establishment of the Oriental River Prawn, Macrobrachium nipponense in Anzali Lagoon, Iran. Aquatic Invasions, 1: Fingerman M Roles of neurotransmitters in regulating reproductive hormone release and gonadal maturation in decapod crustaceans. Invertebrate Reproduction Development, 31: Lepage L, Larson ET, Mayer I, Winberg S Serotonin, but not melatonin, plays a role in shaping dominant subordinate relationships and aggression in rainbow trout. Hormones and Behavior, 48: Meeratana P, Withyachumnarnkul B, Damrongphol P, Wongprasert K, Suseangtham A, Sobhon P Serotonin induces ovarian maturation in giant freshwater prawn broodstock, Macrobrachium rosenbergii de Man. Aquaculture, 260: Mennigen JA, Lado WE, Zamora JM, Duarte-Guterman P, Langlois VS, Metcalfe CD, Chang JP, Moon TW, Trudeau VL Waterborne fluoxetine disrupts the reproductive axis in sexually mature male goldfish, Carassius auratus. Aquatic Toxicology, 100: Mennigen JA, Martyniuk CJ, Crump K, Xiong H, Zhao E, Popesku J, Anisman H, Cossins AR, Xia X, Trudeau VL Effects of fluoxetine on the reproductive axis of female goldfish (Carassius auratus). Physiol Genomics, 35: Nagaraju GPC Reproductive regulators in decapod crustaceans: an overview. The Journal of Experimental Biology, 214: New MB, Valenti WC, Tidwell JH, D Abramo LR, Kutty MN Freshwater prawns: Biology and farming. Blackwell Publishing Ltd. Pp: Peeke HVS, Blank GS, Fliger MH, Chang ES Effects of exogenous serotonin on a motor behavior and shelter competition in juvenile lobsters (Homarus americanus). Journal of Comparative Physiology A, 186: Ruibal C, Soengas JL, Aldegunde M Brain serotonin and the control of food intake in rainbow trout (Oncorhynchus mykiss): effects of changes in plasma glucose levels. Journal of Comparative Physiology A, 188:

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