Evaluation of humpback grouper, Cromileptes altivelis reproduction because of mating between males from the wild and females from cultivation

. There has been a tendency to decrease the genetic quality of aquaculture commodities, which is a significant problem to be addressed immediately. The study aimed to evaluate the reproduction of humpback grouper broodstock resulting from crossbreeding male broodstock from the wild with female broodstock from captivity. This research used 2 (two) concrete tanks 75 m 3 in volume. Each tank was filled with 25 females comprising 15 cultured and 10 broodstock from the wild and conducted from March to November. The broodstock feed consists of fresh fish and squid. The treatment given is the difference in vitamins given. The Tank A was fed an additional vitamin mix, while in Tank B was fed extra vitamins C and E. Recorded parameters such as egg production, egg hatching rate, egg size, spawning frequency, and oil droplet size. The results of humpback grouper reproduction showed that providing fresh trash fish, squid, vitamin C, and vitamin E in the feed yields better results. The spawning frequency was higher in tank B, with 22 times occurrences, compared to tank A, with 17. The highest number of eggs was observed in October, with up to 1,395,000 eggs in Tank B and 1,150,000 in Tank A.


Introduction
Grouper fish have many species, including the humpback or mouse grouper.Humpback grouper is a grouper fish that is very popular with foreign countries and has the highest economic value among other grouper fish.Indonesia exported over 90% of Hong Kong's 165.87 tons of grouper in January 2020 [1].The demand for seafood in Indonesia tends to increase.Despite the decline caused by the Covid-19 pandemic, the demand for grouper has risen again since mid-2020 [2].
The development of humpback grouper hatchery technology is one of the efforts to meet the need for grouper seeds, both in quality and quantity, to support the development of sustainable grouper cultivation activities.In addition, technology transfer is also carried out to be developed in the community.Broodstock availability is one factor that becomes an obstacle in the grouper hatchery business, especially the humpback grouper [3].So far, the main grouper fish spawned come from nature and which is usually caught by fishermen.Obtaining the humpback grouper is relatively difficult because it only exists in certain waters.
To overcome this challenge, it's time to conduct studies and efforts to provide high-quality broodstock for cultivated products [4].Aquaculture is expected to boost the nation's economy and serve as a foreign trade source.Even though the aquaculture sub-sector makes a relatively small contribution to total fish production, its social impact is quite large in driving the economy of rural communities.Therefore, research that leads to breeding is a priority.Thus, the developed marine aquaculture business commodities mainly aim at commodities that have good opportunities in the domestic and international markets, so they are expected to be able to increase the income of fish farmers and support non-oil and gas exports.Humpback grouper has been successfully bred or raised as a consumption fish in floating net cages [5], [6].
Currently, fishermen catch the humpback grouper broodstock from the wild.Producing offspring is vital in supporting aquaculture activities, especially the availability of male and female broodstock [7].One of the factors constraining the grouper hatchery business is the availability of functional broodstock.One of the causes of the difficulty in obtaining broodstock from cultivation is the time it takes to reach sexual maturity.In sustainable fish farming, dependence on broodstock from nature must be reduced gradually and replaced with domesticated hatchery broodstock followed by a selective breeding program [8].
Selective breeding is a program used to improve the genetic quality of the population using selection.If this is accomplished, the subsequent generation will be more valuable.It will grow faster and increase yield, making maintenance more efficient and cheaper.The hope is that these results can be easily cultivated and accepted by the market.Therefore, several studies on breeding have been carried out.The genetic diversity of fish needs to be maintained in the process of using broodstock in hatcheries because gene reduction will result in the loss of some of the genetic characteristics of the offspring [9].This high genetic diversity is also heavily influenced by the number of broodstock in a hatchery population and the number of effective broodstock in a spawning line [10].The broodstock of the first derivative cultivation (F-1), which was used as broodstock for hatcheries, has been successful and has produced F-2 seeds [3], [11], [12].
Furthermore, various fish species have been studied for hybridization to enhance the growth rate, control sex ratios, and generate infertile fish.Besides that, it can also improve meat quality, disease resistance, environmental tolerance, and other properties to make it more profitable [13].Furthermore, the genetic diversity of fish needs to be maintained in the process of using broodstock in hatcheries because gene reduction will result in the loss of some of the genetic characteristics of the offspring.This high genetic diversity is also heavily influenced by the number of broodstock in a hatchery population and the number of effective broodstock in a spawning area [14].

Materials and methods
The individuals used in this study were humpback grouper broodstock from the wild and cultivated broodstock with a weight range between 1.4 -3.2 kg/fish with a total length between 40.0 -52.0 cm.This research was conducted using 2 (two) concrete tanks with a volume of 75 m 3 by an outdoor system.Each tank was filled with 25 female humpback groupers consisting of 15 cultivated humpback grouper broodstock and ten humpback grouper broodstock from the wild.The study was conducted in the Gondol, Bali, Institute for Mariculture Research and Fisheries Extension.
The humpback grouper broodstock was given fresh fish and squid.The treatment given is the difference in vitamins given.The broodstock in tank A was fed with an additional vitamin mix (Vit.B1:0.85%,Vit.B2:0.14%,Vit.E:1.5%, Vit.B6:0.15%,L Ascorbic-Ca:1.82%, PCa:0.3%), while the broodstock in tank B was fed with extra vitamins C and E. The rearing tank is equipped with aeration to provide oxygen while changing the water with running water.The research took nine months, starting in March and concluding in November.The data collected in this study were the production number of eggs, rate of eggs hatching, diameter of eggs, frequency of spawning, and diameter of oil globules.We performed a descriptive analysis of the collected data.

Results and Discussion
The results of observations of spawning in tanks A and B during the study were that the production number of eggs in tank A was 5,384,000, while in tank B, there were 5,648,000 eggs.The hatchability of eggs in the tank A was between 0 -85%, while in the B tank, it was between 0 -90%.In detail, spawning in each tank A and B is shown in Table 1.Furthermore, the diameter of the eggs in Tank A and Tank B is 712-856 and 718-860 mm.The frequency of broodstock spawning of humpback grouper in tub A and tank B was 17 and 22 times, respectively.Some things that need to be considered in the process of gonad maturity to produce seeds include, among others, parent conditions, egg handling, larval rearing techniques, feeding techniques, diseases, and others.Therefore, it must be scrutinized to obtain the expected quality and quantity of seeds.
Based on studies about the spawning of marine fish, it has been discovered that eggs with a lower hatching rate than 40% can negatively impact the development of larvae.This may result in slow development and significant mortality rates before the larvae reach 45 days after hatch.Like a hybrid grouper, this occurs frequently in other marine species [15].In a series of cultivation processes to produce consumption fish, it is necessary, such as nursery to enlargement [16].Nursery is a transitional period to prepare seeds at larger sizes that are more adaptive to cultivation treatments [17].
Grouper nursery is generally carried out in tanks with optimal water sources from the sea.The results of research on gonadal maturity and sex change in groupers.[18] are very important for spawning success.Furthermore, providing optimal feed nutrition is important [19].Therefore, if the feed and environmental conditions are adequate, it can accelerate the vitellogenesis process [20].
The number of eggs from grouper spawning in tanks A and B can be seen in Figure 1 below: Fig. 1.The humpback grouper egg production during the experiment From Figure 1, From April to November, the main humpback grouper in tanks A and B spawned continuously, except in September and March, when there was no spawning.It is suspected that there is a change in weather, so the water temperature is not optimal for spawning.The highest production number of eggs was in October, with 1,395,000 eggs in tank B, while there were 1,150,000 eggs in tank A. The lowest spawning occurred in April, with 105,000 eggs in Tank A and 120,000 eggs in Tank B. During the study, it was suspected that the female humpback grouper had experienced a sex change to male or a transitional period between female and male [4].
Therefore, the success of spawning, which begins with the perfect maturity of the gonads, depends on several factors, including sex change (sex reversal), the feed given, the environment, and others [8].Feed is a very important factor in grouper cultivation [19].Sufficient vitamin content in feed can accelerate the process of oocyte development or "vitellogenesis, [20], [21].The total number of humpback grouper eggs produced during spawning in tanks A and B was not all fertilized eggs, as shown in Figure 2 below the fertilized eggs spawned during the experiment.October, namely 1,010,000 eggs, followed by June, November, and July, respectively, with 800.00, 755.00, and 738.00 eggs.In March and September, there were no fertilized eggs in Tank A and Tank B because spawning did not occur.

Fig. 3.
The number of humpback grouper eggs that were not fertilized during the experiment As a result of observing the number of unfertilized humpback grouper eggs (Figure 3), the number reached 385,000 in October.Then followed in May as many as 377,000 eggs.Several studies related to marine fish spawning and mass production of seeds include yellowfin tuna [22], humpback grouper, [14] hybrid grouper, [23] and many other marine fish.Therefore, the number of fertilized and unfertilized eggs from spawning marine fish, including humpback grouper, fluctuates.

Conclusion
The results indicated that Tank B had the highest spawning frequency, with 22 occurrences, while Tank A had 17.The tanks produced the highest number of eggs in October, with Tank B producing 1,395,000 eggs and Tank A producing 1,150,000 eggs.

Fig. 2 .
Fig. 2. Number of fertilized humpback grouper eggs during the experiment Figure 2 and Figure 3 present results on the number of fertilized eggs and unfertilized eggs observed during the experiment.In rearing tank B, the most fertilized eggs occurred in

Table 1 .
The production number of eggs, rate of eggs hatching, diameter of eggs, frequency of spawning, and diameter of oil globules of Humpback grouper broodstock