Reproductive and growth performances in female giant freshwater prawn following inhibition of gonadal maturation using dopamine and medroxyprogesterone hormone Performa reproduksi dan pertumbuhan pascapenghambatan

One of the main problem in giant river prawn (GFP) culture is early gonadal maturation in female resulting a reduced growth performance. This problem cause economic losses. When GFP at gonadal maturation, somatic growth will be inhibited because energy is used for reproduction. A factorial experimental design using two factors, namely dopamine and medroxyprogesterone, with each factor consist of three levels was applied. Three dopamine levels were 0, 10-5 mol/shrimp, and 10-10 mol/shrimp, while the medroxyprogesterone levels were 0, 75 mg/1.5 mL/bodyweight, and 150 mg/3 mL/bodyweight with a density 15 individual/tank. The utilization of dopamine and medroxyprogesterone in GFP (initial bodyweight : 11.27 ± 0.97 g) through injection at the third periopod was done three times at week 0, 2nd, and 4th with two weeks interval. The results showed that hormone inhibitor treatments affected both growth and reproductive performances in female GFP. The treated individuals showed a lower gonadal maturity indicator values and faster growth rate than control. Gonadal maturity, as shown by gonad histology, in all treatments were lower (previtelogenic and vitellogenic stages) than that in control which is in mature stage. Estradiol concentration premix dopamine 10-10 mol/shrimp and medroxyprogesterone 150 mg/3 mL/ bodyweight treatments are lower than control. In conclusion, dopamine and medroxyprogesterone administration could suppres GSI and gonad development, and also increase growth rate.


INTRODUCTION
Giant freshwater prawn Macrobrachium rosenbergii is freshwater species with high economic value. A rapid gonad maturation on the female broodstock becomes constraint in giant freshwater prawn production. Ra'anan et al. (1991) stated that a female giant freshwater prawn reached the first gonad maturation in 18-26 g. It disrupts the giant prawn production because of the low energy allocation for growth when the gonad is matured. The somatic growth is disrupted since the major energy allocation will be on the reproduction process (Cavalli et al., 2001).
The gonad development of female giant freshwater prawn is naturally affected by several hormone mechanisms (Swetha et al., 2011). Gonad stimulating hormone (GSH) and methyl farnesoate (MF) are the gonadotropin hormones which essentially work in the reproduction glands activity and progesterone in female giant freshwater prawn. The production of GSH and MF are naturally inhibited by the gonad inhibiting hormone (GIH) activity and mandibular organ inhibiting hormone (MOIH) which is produced by X-organ located in the eyestalk (Thongbuakaew et al., 2019).
The crustaceans growth is controlled by the ecdysteroid hormones (molting hormone) located in the Y-organ. The Y-organ will synthesize and secrete ecdysteroids to the growth cells, such as eyestalk and hepatopancreas (Nagaraju, 2011). According to Chang and Mykles (2011), the function of molt-inhibiting hormone (MIH) is to regulate molting process in crustaceans, whereas the crustacea hyperglemic hormone (CHH) provides the carbohydrates and lipids to fulfill the energy requirement of crustaceans (Vinagre & Chung, 2016). The methyl farnesoate stimulates growth in crustaceans. It is supported by Allayie et al. (2010) who stated that the provision of mandibular organ extract was able to rise weight gain in yellowish brown crab Charybdis lucifera.
Hormonal engineering is one of the solution to inhibit gonad maturation and increase growth rate. Accordingly, this study used the hormonal engineering through dopamine and medroxyprogesterone induction as gonad maturation inhibitor in female broodstock. Dopamine is a hydrophilic neurotransmitter located in central nerve system and crustaceans ovary (Tinikul et al., 2009). O'Connell et al. (2013 mentioned that dopamine in vertebrates acts as neurotransmitter which contributes in hypothalamus and pituitary function and also inhibits gonad development. Fingerman (1997) stated that dopamine perfoms to inhibit gonad maturation in prawn through X-organ neuroendocrine cell in terminal medulla in eyestalk to synthesize gonad inhibiting hormone (GIH).
Medroxyprogesterone acetate is a steroid hormone produced by the ovary, adrenal cortex, and placenta in human pregnancy (Suherman, 2008). Medroxyprogesterone is lipophilic (Steele et al., 2013). Daido et al. (2014) mentioned that medroxyprogesterone depressed ovulation in inhibiting hypophysis to secrete luteinizing hormone (LH) and folicle stimulating hormone (FSH). This study aimed to evaluate dopamine and medroxyprogesterone utilization as gonad maturation inhibitor in female broodstock of giant freshwater prawn.

Experimental prawn
The experimental prawn was giant freshwater prawn Asahan strain. The average weight was 11.27 ± 0.97 g. The prawn was four-month old, healthy, no defect, complete body organ, and pathogen-free. Dopamine hydrochloride powder (Sigma Aldrich), medroxyprogesterone, and Estradiol kit (USA) were utilized in laboratory analysis.
The female giant freshwater prawn (initial weight: 11.27 ± 0.97 g) were treated using dopamine and medroxyprogesterone three times on week-0, week-2, and week-4. The dopamine and medroxyprogesterone were injected on the third walking leg. The rearing was managed for three months in a concrete tank sized in 1.5×1×1 m 3 with 15 ind/tank of stocking density. The hormone injection was done in the morning at 7 a.m.. During the rearing, the experimental prawn was fed using commercial feed with 35% of protein content. The feeding rate was 5% of biomass and it was delivered three times a day.

Parameters observation and sample collection Estradiol analysis
Estradiol analysis was managed by collecting 0.3 mL hemolymph sample on the third walking leg using syringe 0.1 mL rinsed using coagulant. The sample was centrifuged and stored in -20°C storage. The estradiol measurement calculation was conducted using ELISA (enzyme-linked immunosorbent assay) method (kit DRG ® Estradiol ELISA (EIA-2693). The estradiol analysis was done at the beginning and at the end of the study.

Gonado somatic index (GSI)
Gonado somatic index (GSI) was observed at the beginning and the end of the study using the following formula (Effendie, 2002) :

Gonad histology
Gonad histology was monitored to observe the gonad microscopic. The method was hematoxyline and eosin staining. It was examined at the beginning and end of the study.

Specific growth rate
The specific growth rate was measured at the end of the study using the following formula (Effendi, 2004) : Table 1. Production and reproduction performance post-gonad maturation inhibition.
Note : Wo = Initial weight (g) Wt = Final weight (g) n = rearing period (day) Survival rate (SR) Survival rate is a percentage of final population compared with initial population. The calculation was done using this following formula (Effendie, 2002) :

Data analysis
The data were analyzed using factorial completely randomized design with two factors, i.e. dopamine and medroxyprogesterone. Data were tabulated using Ms. Excel 2010 and analysis of variance was done using Minitab 16. A significant result would be analyzed further with Tukey test. Gonas histology, survival rate, maturation period, and water quality parameters were described descriptively.

Production and growth performance of postinduction inhibitory
The parameters consisted of estradiol concentration, gonado somatic index, gonad histology, daily growth rate, and survival rate. The result of gonad maturation inhibition using dopamine and medroxyprogesterone presented that D2, M2, and D2M2 were prominent compared with the other treatments (Table 1). It is described by the estradiol concentration and gonado somatic index result. The three particular treatments showed previtellogenic phase and the growth rate was significantly higher than the other treatments.

Gonad histology
Gonad histology was observed in the initial and final period of the study. In the beginning of the study, the gonas histology showed previtellogenic phase (Figure 3), i.e. oval oocyte, nucleolus surrounded by the nucleus, and noticeable follicle cell. The dopamine and medroxyprogesterone treatments presented previtellogenic and vitellogenic phase. On the contrary, the control showed mature phase (Figure 4). On the vitellogenic phase, oocyte appears bigger than previtellogenic phase. There is the nucleolus surrounded by the nucleus, the follicle which surrounded oocyte, and it contains lipid granules. On the contrary, the matured phase, oocyte reached double size compared to the vitellogenic phase and the ovary was full of matured oocyte.
The collection of gonad sample was managed to observe the gonad development. The GSI value at the end of the study showed declining point when dopamine and medroxyprogesterone were delivered. The statement is supported by Daido et al. (2014). Medroxyprogesterone has a certain role to repress ovulation by inhibiting hypophysis to secrete gonad maturation hormone (LH and FSH). Tinikul et al. (2009) stated that dopamine inhibits the gonad maturation process and oocyte development in giant freshwater prawn. On the contrary, Chen et al. (2018) mentioned that dopamine was able to surpress the synthesis and secretion of gonadotropin in teleost. Supporting the later statement, Ciechanowska et al. (2018) also described that dopamine restrains the GnRH biosynthesis process in goat.
The gonad histology at the end of the study explained that dopamine and medroxyprogesterone were in the previtellogenic and vitellogenic phase. It can be seen from Figure 4 that several oogonia cells were in the early stage and developed oocytes were spotted because of the cytoplasm development compared to the control treatment which the nucleus was fused. According to the gonad histology by Ngernsoungnern et al. (2009), in the previtellogenis phase, oocyte was on the oval-shaped, nucleolus was surrounded by nucleus, and follicle cell was spotted. Compared to the vitellogenic phase, oocyte appeared bigger than the previtellogenic phase, nucleolus was surrounded by the nucleus as well, and there were fatty follicle cell surrounding the oocyte. The control treatment presented bigger oocyte in the vitellogenic phase and ovarium was full of matured oocyte. The particular character was considered as matured individual (Soonklang et al., 2012;Kankuan et al., 2017).
The specific growth rate in the D2 treatment (dopamine 10 -10 mol) was higher that the others. It was preassumed that a low dosage of dopamine was adequately effective to boost a higher growth. According to Jin and Hashizume (2014), dopamine is involved in growth hormone and prolactin regulation mechanism in goat. Dopamine restrained the somatostatin hormone in hypothalamus, thus the growth hormone releasing hormone (GHRH) was able to be stimulated to secrete growth hormone.
The declined survival rate at the end of the study was presumably caused by stress condition during the injection. Stress affects the immune system through metabolic mechanism (Yeh et al., 2010 ;Leland et al., 2013). Chang et al. (2007) stated that two hours after dopamine injection in dosage 10 -6 , 10 -7 , and 10 -8 mol, the oxyhemocyanin decreased significantly. Oxyhemocyanin is a blue pigment formed by the oxygen and hemocyanin in ratio 1:2 (Cheng et al., 2013), while hemocyanin is a glycoprotein contained copper and it is usually found in the hemolymph (Zheng et al., 2016). The declined oxyhemocynanin disrupted the metabolism, osmoregulation, and resporation system which later caused stress and unable to adapt to the environment (Chang et al., 2016). Adding the previous statement, Camacho-Jimenez et al. (2017) reported that a 2 × 10 -6 mol of dopamine potentially controlled the osmoregulation system in the whiteleg shrimp Litopenaeus vannamei. Osmoregulation is a homeostatis system to maintain the milieu intérieur stability through osmotic balance regulation amongst intracell and extracell (Maghfiroh et al., 2019).
Molting is the detachment of older cuticula and forms a new cuticula layer (Rocha et al., 2012). The molting process is usually followed by the length, weight, and width changes (Fujaya et al., 2011). In crustaceans, molting is triggered by several factors, i.e. growth, reproduction, and stress (Hess, 2014). The giant freshwater prawn is a cannibal (Mendler et al., 2015). When molting occurs, it lacks of strength. It provokes the stronger and not-experiencing-molting individual to attack them. This leads to death for those which experiencing molting at a certain time (Mendler et al., 2015).