Effect of Pregnant Mare Serum Gonadotropin and Antidopamine on the gonads of female bileh fish (Rasbora maninjau)

Bileh fish (Rasbora maninjau) still have difficulty spawning in a controlled manner in aquaculture containers because the maturation of female fish gonads is slow and takes a long time. Internal factors, including the hormonal system, strongly influence the maturation of fish gonads. This study aims to determine the effect of Pregnant Mare Serum Gonadotropin (PMSG) and antidopamine on female gonads of bileh fish. The research used an experiment with a CRD (Completely Randomized Design) experimental design. The experiment consisted of four treatments and three repetitions. The treatments were the administration of PMSG and antidopamine hormones at different doses through feed, namely: 0 ml/kg feed (P1), 0.5 ml/kg feed (P2), 1 ml/kg feed (P3) and 2 ml/kg feed (P4). The research procedures included container and fish preparation, hormone, and feed preparation, fish rearing, and data collection. The test parameters evaluated were weight gain (Wg), gonadosomatic index (GSI), and gonadal maturity level (GML). Data on test parameters were analyzed with statistics (Anova). The research results showed that the administration of PMSG hormones and antidopamine through feed had a significant effect on weight gain, gonadosomatic index and gonad maturity level of bileh fish (P<0.05). Effective and best treatment at P3 (1 ml/kg feed) with Wg 0.22grams, GSI 8.58%, and GML IV 60%.


Introduction
Fish biodiversity in the waters of Sumatra island is high, and one of them is bileh fish from the genus Rasbora.Reported fish species of Rasbora members found in Sumatra island waters include Rasbora api, Rasbora kluetensis, Rasbora nodulosa, Rasbora truncate [1], Rasbora arundinata, Rasbora bindumatoga, Rasbora maninjau, Rasbora haru [2], and Rasbora tawarensis endemic to freshwater lakes [3].Rasbora is a freshwater fish found in rivers, lakes, puddles, or rice fields.Bileh fish is a local name for the people of West Aceh and Nagan Raya districts.Bileh fish belongs to the Rasbora maninjau species [4].
The fulfillment of bileh fish so far still relies on catches from nature, reducing the population of fish in nature.Overfishing must be reduced, and efforts must be made to cultivate bileh fish for the community's needs.Researchers have studied the domestication of bileh fish in aquaculture containers [5], type of feed, and growth [6,7,8,9].Bileh fish reared in aquaculture containers experience problems in gonad maturation.Internal and external factors influence the process of gonad development.Internal factors are in the form of hormone performance, and external factors are in environmental conditions.Fish will respond to environmental factors through hormonal regulation, which is connected between the brain to the hypothalamus, then to the pituitary, and the gonads [10].Environmental signals will be received by the central nervous system (brain) and forwarded to the hypothalamus.The nerves of the fish brain receive environmental signals, then sent to the hypothalamus.The hypothalamus responds by secreting GnRH (gonadotropin-releasing hormone) and dopamine.Environmental signals will indicate that dopamine acting on the pituitary gland will inhibit GnRH.Then, gonadotropin hormones FSH (Follicle Stimulating Hormone) and LH (Luteinizing Hormone) that act on the target organs of fish gonads.FSH stimulates the vitellogenesis process, while LH stimulates the gonadal maturation process until ovulation.Therefore, hormonal manipulation is one of the fastest ways in gonadal maturation.
Pregnant mare serum gonadotropin (PMSG) is a hormone commonly applied to farmed fish.PMSG hormone contains elements of FSH and LH.PMSG hormone administration must be combined with antidopamine (AD).Antidopamine is a chemical substance that can stop the work of dopamine, as a substance that inhibits the activity of releasing the hormone GnRH from the hypothalamus.Dopamine works to inhibit gonadal maturation by acting as a gonadotropin-release inhibiting factor (GnRIH).With the presence of antidopamine, the neurotransmitter is expected not to inhibit gonadal maturation so that the gonadal maturation process can be achieved more quickly.The combination of PMSG and antidopamine hormones has a positive effect on the reproductive performance of Tor douronensis, Channa striata, Poropuntius tawarensis, and Osteochilus hasselti [11,12,13,14,15].The combination of PMSG and antidopamine hormones is expected to provide optimal results in the gonad maturation of bileh fish.The research objective was to analyze the effect of PMSG and antidopamine on the gonads of female bileh fish.

Research Design
The research was conducted in the Laboratory of Aquaculture Science and Environment of Teuku Umar University from September to October 2023.A completely randomized design (CRD) was used in this research.Four treatments were given with three replications.The treatments given were PMSG and antidopamine with the trademark Oodev.The Oodev treatment was added to the feed at various doses.The treatment doses given are: Treatment 1 (P1) = 0ml/kg feed (control) Treatment 2 (P2) = 0.5ml/kg feed Treatment 3 (P3) = 1ml/kg feed Treatment 4 (P4) = 2ml/kg feed

Research Procedures a. Preparation of fish containers
Fish were kept in aquarium containers of 60x40x40 cm.Aerators are installed in all rearing containers to supply the fish's oxygen needs.The aquarium is cleaned first by washing and sterilizing using a disinfectant.After the aquarium is clean and dry, the water is filled to a height of 25 cm.The water used for fish rearing is precipitated first for at least 24 hours.Deposition is carried out to separate particles carried by water.With gravity, the particles will settle to the bottom of the tub.

b. Fish preparation
The fish used in this research is bileh fish (rasbora), obtained at UPR Mina Mandiri, Beutong, Nagan Raya, Aceh.There were 120 fish used for the experiment.Each rearing aquarium was filled with ten fish.The size of the fish used had an average fish weight of 2.02 ± 0.64 grams/fish and an average fish length of 6.17 ± 0.59 cm/fish.

c. Mixing hormones in the feed
The addition of hormones in feed begins with mixing oodev hormone with aquades and egg white.Hormone oodev is added according to the treatment dose (0, 0.5, 1, and 2 ml/kg feed).Aquades 5 ml is added for hormone dilution, while egg white (10 ml) is added to prevent leaching.The hormone mixture is stirred evenly and then put into a spray bottle.Spray the hormone onto the feed while stirring evenly.Air dry the feed and feed it to the fish.

d. Bileh fish rearing
Fish were reared for 42 days and given feed containing hormones according to the treatment.The feeding frequency was given three times daily in the morning, afternoon, and evening at satiation.Water quality was controlled during maintenance, and water quality parameters, including temperature, DO, and pH, were measured.Fish feces in the aquarium were cleaned, and the water was changed by 30-50% if the aquarium water was cloudy.

e. Data collection
Observations and data collection were conducted on the first day of the study (initial), every 7 days and on day 42 (final).The data required include brood weight gain, gonadosomatic index and gonadal maturity level.All data were written in a research logbook and analysed after completion of the study.Note: Bg= Gonad weight (grams), Bt= Fish body weight without gonads (grams)

Gonad Maturity Level (GML)
The fish were dissected, and their gonads were visually observed based on the GML table (table 1).Description of fish gonads by making gonad histology preparations using the paraffin method and Hematoxylin Eosin (HE) staining.The procedure for making histology preparations refers to previous research [16].Table 1.Fish Gonad Maturity Level (GML) Criteria based on Syarif's research [17].

I
Immature A small ovary is 1/3 of the body cavity and has an oval egg shape, ovary color pink and transparent.
Small eggs, invisible to the naked eye, 1-16 µm in diameter and transparent.

II
Maturing The ovary is a small 1/2 of the body cavity, elongated.Ovary color pink, transparent.
Eggs invisible to the naked eye, clear eggs, size 10-21µm in diameter.

III Maturing Ripe
Small ovary 1/2-2/3 of the body cavity, right and left gonads not symmetrical.Ovary color yellow, granules visible, blood vessels visible on the surface.

Ripe
Ovaries 2/3 to full in body cavity, orange-pink color, blood vessels visible on the surface.

Data analysis
Data on weight gain (Wg) and gonadosomatic index (IGS) were analyzed statistically (Analysis of Variance) using SPSS 21.0.Duncan's further test was conducted if the treatment results showed a significant effect (P < 0.05).Data on the level of gonadal maturity (GML) were presented in figures and tables, which were then described descriptively.

Ethical statements
This animal study was approved by Teuku Umar University ethics committee.This research was carried out following animal welfare standards and policies according to the Government Regulation Concerning Animal Welfare, Chapter III Animal Welfare, Part 1; Article 83.

Results and Discussion
Fish weight gain during rearing is presented in Figure 1.Fish weight growth increased in all treatments and showed positive results.The growth value in the P4 and P3 treatments was higher than in the P2 and P1 treatments.Based on statistical tests, feeding containing PMSG + antidopamine hormone with different doses significantly affects the growth of bileh fish weight (P < 0.05).Fish growth occurs when the nutritional needs in the feed are met, and the fish live in an appropriate environment [17].During the study, the provision of PMSG + Antidopamine hormones in feed affects the weight growth of bileh fish.Fish body weight gain is caused by the influence of meals, gonad development, and fat deposits [18].Growth in prospective broodstock fish increases due to gonad development [19].The increase in gonad weight goes hand in hand with the IGS value, the greater the gonad weight, the greater the IGS value [13].P4 (2 ml/kg feed) Gonadosomatic Index (%) treatment dose of 2 ml/kg feed.Based on statistical tests, feeding containing PMSG + antidopamine hormone with different doses significantly affected the GSI of bileh fish (P < 0.05).Duncan's further test, treatment P4, significantly differed from treatments P1, P2, and P3.The GSI value increases with the increase in hormone dose (P1, P2, P3), and in P1 (control), the GSI value is low because it is without PMSG hormone administration.The difference in the GSI value of bileh fish is caused by oocyte development, which is influenced by the PMSG hormone in the feed.The increased IGS value indicates the growth of oocytes filled with vitellogenin [13].In the vitellogenesis process, the oocyte volume enlarges, and the increase in GSI value aligns with the rise in fish gonad volume [17].The vitellogenesis process is responsible for fish's high and low GSI values [20].Administration of PMSG + antidopamine can provide hormonal stimulation of early gonadal development of bileh fish (Figure 2).PMSG has biological properties such as luteinizing hormone (LH) and follicle-stimulating hormone (FSH).FSH plays a role in the process of egg formation in fish, while IOP Publishing doi:10.1088/1755-1315/1329/1/0120096 LH plays a role in stimulating the process of gonad maturation.The results of histological observations of the gonads, feed containing hormones can stimulate the development of gonads faster, compared to without hormone administration (control/P1).The pattern of oocyte development is characterized by the position of the nucleus, which begins to move to the edge, and there is no vesicle in the cytoplasm.Ovaries began to develop an increase in yolk granules as a sign of the beginning of the previtellogenesis phase [21].Based on observations according to Table 1, the level of gonad maturity (TKG) of bileh fish in each treatment varies.In P1 the fish were at TKG III as much as 30%, P2 fish were at TKG IV as much as 30%, P3 and P4 fish were at TKG IV as much as 60%.The difference in TKG in each treatment is caused by the effect of hormones in accelerating gonad development.TKG results are in accordance with the GSI value, where the highest GSI value is found in P3 and P4, which produce the highest TKG IV, as much as 60%.Seluang fish (Rasbora einthovenii) had 50% TKG IV [17] and silver rasbora produced 70% TKG IV [20].Internal and external factors greatly influence the TKG value.External factors include temperature, current, the presence of the opposite sex, and others.Internal factors include species differences, age, and other physiological traits [22].

Conclusion
Administration of PMSG and antidopamine hormones through feed significantly affected broodstock weight gain (Wg), gonadosomatic index (GSI), and gonadal maturity level (GML) of bileh fish (P<0.05).PMSG and antidopamine hormones can increase the GSI value of bileh fish and be used as a stimulus for early-stage gonadal maturation.The treatment is effective and efficient in the P3 treatment (1ml/kg feed) with a Wg value of 0.22 grams, GSI 8.58%, and GML IV 60%.

Figure 1 .
Figure 1.Weight gain of bileh fish during rearingThe gonadosomatic index (GSI) value of bileh fish during maintenance is presented in Figure2.The GSI value obtained ranged from 3.81% to 8.89%.The highest value was found in P4 with the

Figure 2 .
Figure 2. Gonadosomatic Index value of bileh fish during rearing

Table 2 .
Percentage of gonad maturity level (GML) of female bileh fish after being given PMSG+AD n = 10 individuals