Embryonic and Larval Development of Hybrid Giant Grouper × Potato Grouper (PGGG) Male and Tiger Grouper (TG) Female

This study aims to collect data and information of embryonic and larval development on the cross breeding between hybrid Giant Grouper × Potato Grouper (PGGG) male and Tiger Grouper (TG). The body weight of broodfish above 20 kg for male and 4 kg for female. Broodstock selection of Hybrid Giant Grouper × Potato Grouper (PGGG) male can usually be stripped by applying gentle pressure to the abdomen between the pelvic and the vent fins. Induced spawning by mixing sperm and eggs manually. Signs of a mature gonadal female are characterized by an enlarged abdomen, slow movements and tend to tilt, the genital opening increasingly swollen and red in colour. The results show that spawning at night 23.30 with an average egg diameter of 0.85±0.09 mm. The eggs hatch at 15.45 or 16 hours 15 minutes. Hatching rate being 24%-32%.


Introduction
Hybridization has been carried out on various fish species with the aim of increasing growth rates, manipulating sex ratios, improving meat quality, increasing disease resistance, increasing tolerance to the environment, and having other superior properties [1].Until now, there are several types of hybrid grouper that have been produced, among which are crosses between Epinephelus coioides, E. corallicola, Cromileptes altivelis, E. polyphekadion and E. akaara .In the grouper farming industry, hybrid grouper is a fish that has popularity as in TGGG hybrid fish, which is a cross between tiger grouper (Epinephelus fuscoguttatus) TG and kertang grouper (E.lanceolatus) GG, because it has fast growth with high economic value.TGGG hybrid grouper is often referred to as the most successful hybrid combination, as it is able to grow quickly with high survival [2; 3] and able to tolerate a wide range of environmental parameters [3].1328 (2024) 012020 IOP Publishing doi:10.1088/1755-1315/1328/1/012020 2 At the Marine Aquaculture Research Center, Gondol, Bali, hybridization has been carried out between kertang grouper (GG) and potato grouper (PG) which produces offspring that have high growth.This breed (PGGG) has become a functional male and female parent after being reared for six years.This is in line with Luin et al. (2013) who studied sexual maturation and gonadal development from TGGG, and showed that TGGG hybrids began to grow as females and males.In 2016, fertilized eggs were found in a hybrid TGGG brood tank.The purpose of this study was to obtain data and information on the embryological development and growth of hybridized larvae between male Hybrid Giant Grouper X Potato Grouper (PGGG) and Female Tiger Grouper Fish (TG).

Ethical Approval
This research procedure was in accordance with the principles of animal welfare use and principles.This research procedure was in accordance with the principles of animal welfare use and principles.There are no animals harmed or improperly treated during this research.The test animals in this research were treated properly according to the optimal environment, both physical and chemical oceanographic parameters.

Preparation of Tiger Grouper Female
Selection of female Tiger Grouper that are ready to spawn or mature gonads weighing at least 4 kg.Females who are ready to spawn or mature gonads are characterized by a enlarging abdomen, swollen genital opening (cloaca) and reddening.The female is declared ready to spawn, shown when checking by cannulation there are eggs that have separated or not clotted and are relatively large with an egg diameter of 450 μ-500 μ, then the female is ready for hormone injection.Injecting using HCG hormone at a dose of 250 IU / kg.

Preparation of Hybrid Giant Grouper (PGGG) Male
The broodfish selected to get a male is weighing minimal 20 kg.Selection of male Hybrid Giant Grouper × Potato Grouper (PGGG) by striping on the belly of the fish.After selection of the male, then it is given an anesthetic will be calm and does not rebel at sperm collection time's.Sperm fluid is accommodated in a container, and must not mix with sea water or urine from the broodstock, then put into refrigerator.

Hybridization
Hybridization in Tiger X Grouper by artificial mating (intensive spawning), where fish spawning that occurs is given hormonal stimulation to accelerate gonad maturation, and the ovulation process is carried out artificially, namely by stripping techniques.Checking female tiger grouper fish starts at 22.00.When checking, the tank water is lowered to a height of 30 cm.Signs that the female is ready or has mature gonads are characterized by an increasingly enlarged stomach, slow movements and tending to tilt, the genital opening becomes increasingly swollen and red, the body color, especially the white gills, becomes pale.Then the female mother is cannulated on the genitive part to see the condition of the eggs.Eggs that are ready for artificial mating are characterized by an enlarged egg diameter (750μ-800μ), the color is clear and when put into floating seawater or at least floating.The artificial mating process begins by massaging the abdomen of the female mother of Tiger Grouper slowly, after the eggs come out and are accommodated in a basin, spray Giant Grouper sperm at a dose of one milli liter of sperm for one million E. fuscogutattus eggs.Then stirred using chicken feathers so that sperm is homogeny.The process of stirring sperm with eggs lasts about 10-15 minutes, after that leave or let the eggs stand for about 5-10 minutes so that the fertilization process takes place perfectly.The next process is that the eggs are put into an incubation tub equipped with running water and aeration

Larva Rearing
The fertilized eggs are then put into the larval tub.The larval rearing technique is carried out by following the tiger grouper and batik grouper maintenance techniques.Feeding the larvae until they become juvenile is adjusted to the development of their mouth openings.The feed given is in the form of natural feed and artificial feed Feeding patterns and water management can be seen in Table 1 below The parameters observed in this study include egg development, hatching rate (HR), larval to juvenile growth, and survival rate (SR).In addition, the percentage and type of deformity are observed at the end of each cycle

Egg Development Stages
The initial process of embryonic development of GGPG X TG hybrid grouper is fertilization, which is when the egg and sperm cells that form the zygote.This spawning process occurs in the evening at 23.30.The results of measuring eggs with an average diameter of 0.85 ± 0.09 mm, with the characteristics of being round, transparent, and floating in water.Cleavage takes place after fertilization, where division I in GGPG X TG hybrid grouper occurs at 23.45 or after a time interval of 15 minutes hours after hatching (hAF) has resulted in a zygote that divides into two cells.The division of two cells begins with the formation of a straight line at the center of the blastomere which then shrinks and then divides into two cells with the same cell size (Figure 1).

Larval Development
The morphological characters of larval and juvenile development are described in Figure 2. The results of the observation found that larvae aged 0 days (D-0) with the yolk are still full and prospective eyes have formed.The long development of larvae during the study can be seen in Figure 3.The total length of D-1 larvae averages 2.48 mm, the body looks transparent, the digestive tract looks straight like a tube while the mouth and anus are still closed.On D-1 egg yolk remains 80%, eye pigmentation is increasingly pronounced, the digestive tract is tubular straight.In D-3 egg yolk remaining 10% eye pigmentation is already functioning.In D-5 the organs appear to be more perfect.At the age of 7 days the spines of the upper and lower fins begin to grow.The pectoral fin is the first appendicular structure to develop, followed by the spiny dorsal fin and the spiny ventral fin, almost simultaneously, and then by the caudal fin, soft dorsal and soft anal simultaneously, and finally the spiny anal.Visual observations showed that most larvae swim actively during the day and at night form swarms near the edges of the tank.In larvae aged 16 days, the upper fin spines and lower fin spines are increasingly elongated and will shorten or break off before the larvae become juvenile, as well as the digestive organs and internal organs of this hybrid grouper larva are complete and resemble adult fish so that feeding other than rotifers, needs to be given artemia and artificial feed (Figure 3).

Hemotology Profile
The Hb content in the blood of of GGPG X TG hybrid grouper larvae is 6.85 g/dL (Table 3).Meanwhile, the hematocrit value is 30.00%.The number of erythrocytes was 1,785,000 cells/mL, while the number of leukocytes was 283 cells/mL.

Embryology and Larva Development
The newly hatched Plectropomus leopardus larvae have a yolk volume of 1.23 x 10 -1 mm 3 and oil globules volume of 4.9x10 -3 mm 3 [4].Otherwise, eye pigmentation has formed although it is still faint, otoliths are visible (Figure 1A).Otoliths are useful for balance in swimming and to determine the age of fish [5].Larva D-1 still have approximately 80% yolk remaining, eye pigmentation appears increasingly clear, the digestive tract appears in the shape of a straight tube (Figure 1B).Larva of Epinephelus fuscogutattus at first day have a straight tube-shaped digestive tract without curves (villi) [6].Swim bubbles have not yet been formed so that the larvae are in a floating condition following the existing media in maintenance so that aeration needs to be reduced.Meanwhile, larvae at second days still have approximately 10% yolk remaining, eye pigmentation has occurred, the eyes are starting to function, and the mouth opening is visible.It's relevant that the size of the mouth opening of Plectropomus leopardus larvae after the yolk runs out is 145-150 microns so the suitable food is copepod naupli [7].The swim bladder begins to appear, the foregut is visible which will later turn into the stomach.So that at that time the rearing medium needs to be given plankton with the intention of providing conditioning to the media water, while in the afternoon small rotifers (SS) are given, namely 120-140 mm which are given together with copepod naupli in the hope that the larvae will prey on the rotifers and copepod naupli so that larvae will get nutrition from outside.
Larva at 3 days have the boundaries of the front and back intestines as well as the rectum are clearly visible, the villi are not yet visible or are still thin, the mouth opening is normal, the eyes are functioning, and the swim bladder appears increasingly clear (Figure 2A).In the physostome-shaped swim bladder (which is connected to the digestive tract) the filling of the swim bladder is carried out for the first time along with the pigmentation of the eyes and the mouth opening of the larvae [5].In Coastal snapper larvae, the swim bladder is filled for the first time when the larva begins to eat food from outside.So at that time the larvae are expected to start moving and prey on the food in front of them [8].
Larva at 5 days have eyes functioning normally, as is the swim bladder, and the mouth is functioning to prey on food in front of it (Figure 2B).However, for larvae 4 days, this is the critical period for the larvae because the supply of nutrients from the inside or egg yolk has run out so the larvae need nutrition from the outside.For this reason, it is necessary to control the contents of the stomach, namely by squeezing the larva's stomach and looking at it under a microscope.If the larvae eat, biota can be seen in their stomach.If they don't eat, their size will decrease and eventually many will die.During metamorphosis, spines are gradually reabsorbed, pigmentation patterns are formed, habitat changes from pelagic to bottom dwellers, and weaning becomes possible [9].The metamorphosis process in groupers takes more than two weeks to complete and the larvae are known to be very sensitive to stress at this stage.Until metamorphosis is complete at 25 to 50 days, high mortality rates are usually experienced.We also consider stress due to environmental conditions (especially light and temperature) as another factor of death [10].
The spins of humpack grouper larvae begin to appear at 8 days, but the spins are generally clearly visible at 12 days after hatching.Different, the fish larvae of E. fuscoguttatus, spins began to appear as early as 9 days after hatching [11], larvae of L. argentimaculatus at 5 days [12], and larvae of tiger grouper E. fuscoguttatus x giant gouper E. lanceolatus spin at 10 days, it is indicating their strong swimming capabilities at early stages [3].The humpback grouper larvae spin reaches its maximum length when the larvae at 21 to 30 days [13] [3], while the spin of E. fuscoguttatus fish larvae began to reduce after 20 days [3].

Water Quality
Water quality is a critical factor in the success of fish hatchery activities, especially for the survival and health of fish.Optimal water temperatures vary between species but must be maintained within a specific range to ensure proper development and survival of fish eggs and larvae.Temperature that grouper farmers can culture TGGG hybrid grouper between 26°C and 30°C for optimal growth and condition [14].
The salinity levels must be carefully managed to match the natural requirements of the species being cultured.Both the eggs of TGGG hybrids and tiger groupers have a wide range of salinity tolerance from 25 to 35 parts per thousand (ppt).Sub-optimal salinity levels led to a large rise in the prevalence of abnormal larvae, indicating that keeping incubation at 30 ppt can greatly lower the risk of abnormalities in larvae, which is important for the health and survival of fish raised in hatcheries.[15]

Hematology Profile
The Hb level in Cromileptes altivelis weighing 25.49 ± 0.78 g was 4.1-5.The amount of hemoglobin is generally directly proportional to the number of erythrocytes.The low concentration of hemoglobin indicates the occurrence of anemia in the body of the fish.Fish suffering from anemia have a low concentration of hemoglobin due to a decrease in the number of erythrocytes.Hemoglobin levels in the blood related to the balance of blood plasma osmolarity [16].
Normal hematocrit values ranged from 27.3-37.8%.Hematocrit values in teleost fish range from 20-30% and in some marine fish species around 42% [17].The results of the hematocrit examination can be used as a benchmark to determine the health condition of the fish.Salmon (Salmo salar) hematocrit values can vary from 44 to 49%, depending on a number of parameters including body weight, temperature, fish strain, and food [18] [19].A decrease in hematocrit values can be influenced by changes in environmental conditions or environmental pollution which makes fish stressed.Hematocrit es the ratio between red blood cells and blood plasma which affects red blood cells.Hematocrites can be used as an indicator to show the health condition of fish.The number of hematocrites in the blood fluctuates.The increase in hematocrit levels can be influenced by two factors, firstly changes in environmental parameters and the physiological state of the fish.A low hematocrit number also indicates contamination [20], lack of food, low feed protein content, vitamin deficiency or as an indicator of pathogen infection [16] [18].

Conclusion
Larva D-0 still have full of yolk, the eyes is already formed.Then larva D-1 yolk remain 80%, eye pigmentation is increasingly pronounced, and the digestive tract is straight tubular.Larva D-2 yolk still 10%, eye pigmentation is functioning and swimming bubbles are visible.Then, spin begin to appear on days 8 to 12 after hatching and reaches a maximum length of 25 to 30 after hatching.

Figure 3 .
Figure 3. Growth of GGPG X TG hybrid grouper larvae

Table 1 .
. Feeding patterns and water management of larval rearing.

Table 2 .
Water quality parameters of GGPG X TG hybrid grouper larvae hatchery trough

Table 3 .
Hematology Profile of GGPG X TG hybrid grouper larvae