Abstract: Background and Objective: The giant gourami (Osphronemus goramy; Lacepede, 1801) is one of Indonesia's main freshwater fish of economic benefit for food security. This study evaluated the effects of vitamin C dosages used for immersion embryonic and larval of giant gourami. Materials and Methods: The vitamin C using Ascorbic acid standard (FLUKA, Sigma-Aldrich), each weighed 50, 100, 150 and 200 mg and dissolved in 1 L of fresh borehole water is referred to as P2, P3, P4 and P5. At the same time, 1 L of fresh borehole water not added with vitamin C is called P1 (Placebo). Each treatment was formulated with 5 L and poured into an incubator tank with a capacity of 10 L Then, as many as 150 eggs are incubating per experiment in each incubator tank. These larvae of each experiment were rearing for 20 days using live feed (tubifex) 40% per feed. Each treatment was three replicates. Results: In this study, larval hatching rates varied from 78.59-98.22%, with the survival of endogenous feeding periods ranging between 93.08 and 99.10%. The specific growth rate increased from 11.14 and 13.38% per day. The Coefficient of thermal growth increased from 1.49-1.70. Larval mortality after exogenous feeding for 20 days of the experiment decreased from 21.11-5.56%. The Coefficient of variation in weight was between 3.19 and 14.04%. Conclusion: Giant gourami eggs and larvae hatched and survived and grew well in immersion with a dose of vitamin C up to 200 mg L–1.
INTRODUCTION
The aquaculture sectors play is an essential aspect in Indonesia’s economic direct income-generating activities, livelihoods diversity, the contribution of animal protein and foreign trade income. The giant gourami (Osphronemus goramy; Lacepede, 1801) belong to Indonesia's major freshwater fish of economic values. In this nation, earthen freshwater pond aquaculture of giant gourami is a very early practice1-3. The dependability of giant gourami of fish farmers on fry availability is a limiting factor in increasing production4. Therefore, embryo development must be was standardized and widely practised for quality larval production giant gourami. The main factors, which directly affect the embryo development, hatching rate and survival of larval are water quality5-8. The age and size of broodfish are also affected by hatching rate9,10, supplemented vitamin C in the diets broodfish11 and tank background6,12-14, including stocking density15-17. Among these factors, water quality enriched formulated with mineral and vitamin significantly affects larval’s hatching rate and survival7,11,18,19. Nevertheless, several authors have reported used vitamin C for egg immersion and larval development11,20,21. Based on the search for references, we have not found immersion of embryo with a vitamin C the giant gourami egg hatching media, including thermal growth coefficient, Fulton's condition factor and yield.
We hypothesized that egg immersion of giant gourami with different dosages of vitamin C could improve hatching rate, growth and larval survival.
Therefore, the objective of the current study was to evaluate the giant gourami egg immersion effect with vitamin C different dosages to the egg incubation media on the hatching rate, growth performance and larval mortality of these commercially crucial giant gouramis during the early life stage. This finding is an immense significance to overcome the gap in knowledge about hatching Giant gourami eggs.
MATERIALS AND METHODS
Study area: This study was carried out at Aquaculture Laboratory, Department of Aquaculture Faculty of Fisheries and Marine, Universitas Riau, Indonesia from February to April, 2020.
Stock solution of vitamin C: The vitamin C or Ascorbic acid using Ascorbic acid standard (FLUKA, Sigma- Aldrich), each weighed 250, 500, 750 and 1000 mg and dissolved in 5 L of fresh borehole water (the dosages were 50, 100, 150 and 200 mg L–1) is referred to as P2, P3, P4 and P5. At the same time, 5 L fresh borehole water which did not add vitamin C is called P1 (Placebo). The P1, P2, P3, P4 and P5 formulated poured into tank incubators with an inlet diameter of 18 cm, each incubator capacity 10 L.
Experimental design: Two thousand two hundred fifty eggs naturally spawning results from giant gourami collected from fish farmers at Sawah Baru townlet, Kampar Regency of Riau Province. After that, all eggs we carry out by car for a long time of 30 min to the Aquaculture Laboratory, Faculty of Fisheries and Marine, Universitas Riau, Indonesia. We carry out egg disinfection before transfer to the yolk sac rearing tank. Immersion in 200 ppm for 1 min peroxyacetic is using in this experiment. Furthermore, as many as 150 eggs were incubating in each incubator tank. Each treatment was three replicated. During egg incubation, we carry out a continuous aeration process.
Twenty-five eggs were taken at random at 20 hrs after the broodfish spawn in each trial to determine the Fertility Rate (FR). We calculated Hatching Rates (HR) at 42 hrs after brood fish spawn. After the eggs hatch, to raise larvae, a using 0.27 m3 (30×30×30 cm) tank with a capacity of 15 L Larvae were stocked with 30 fish/tank. The average initial weight and length were 13.6 mg and 9.1 mm, respectively. Larvae were stocked in five trials, continuing the egg hatching experiment. Eight-day-old larvae, we are rearing for 20 days using live feed (tubifex) 40% per feed with a frequency of three times a day at 08.00, 13.00 and 18.00. Every day the dead larvae were recorded to estimate the survival rate. The Coefficient of thermal growth is calculated based on the formulate Jobling22. Larval growth parameters such as total length (mm) and wet weight (mg) were measured using a measuring ruler (mm) and digital electronic weighing 0.01 mg (OHAUS, Model CT 1200-S, USA), respectively. The parameters measured use analyzed with the following Eq:
Water quality: This study monitored water quality parameters such as temperature, dissolved oxygen and pH in an egg incubation tank and nursed larvae in tanks. They monitored water temperature, dissolved oxygen and pH daily. The measured water temperature with a thermometer (Celsius scale). The determined pH values with a pH meter (digital mini pH meter, 14 pH, IQ Scientific, Chemo-science Thailand Co. Ltd., Thailand). An oxygen meter (YSI model 52, Yellow Spring Instrument Co., Yellow Springs, OH, USA). Total alkalinity, total hardness and nitrate monitored were recorded twice a week for all the tanks by standard methodology is presented by Boyd et al.23.
Data analyzed: SPSS software (version 16.0 for Windows; SPSS Inc., Chicago, IL) use for data analysis. Kolmogorov-Smirnov statistics were used to test data normality. We used Levine's test to examine the absolute residuals from homogeneity. One-way ANOVA was used to analyze the effect of each treatment, followed by post hoc Duncan's multiple range tests24. The 95% confidence level (p<0.05) was the threshold to identify significant differences. Standard deviation (±SD) complete all means data. The figures were plotting using Microsoft Excel professional plus 2019.
RESULTS
Water quality parameters: We recorded the value of water quality in the tank during embryonic development and larval rearing. The water temperature in the embryones incubation and larval nursed ranges from 27.0 and 29.0oC. The dissolved oxygen fluctuated between 6.2 and 6.4 mg L–1 and the pH was 6.1 and 6.7. Total alkalinity was 60 and 66 mg L–1 as CaCO3, hardness is varied 58 and 70 mg L–1 as CaCO3, while Nitrite-Nitrogen (NO2-N) exceeded 0.01 and 0.03 mg L–1 (Table 1).
Biometric performance: In Table 2 represents the fertility rate, hatching rate, survival endogenous feeding period, growth rate data, yield, mortality and coefficient variation of length and growth. The larvae hatching rate varied from 78.59 and 94.62%, with survival endogenous feeding periods ranging from 93.08 and 99.10%. On the other hand, the specific growth rates went from 11.14 and 13.38% per days, whereas thermal growth coefficients were between 33.99 and 57.78. Larvae mortality after exogenous feeding period for 20 days experiment ranged between 5.56 and 21.11%, with Coefficient of variation of weight varied from 3.19 and 14.04%.
| Table 1: Water quality parameters in an egg incubation tank and larvae reared | ||||
| Embryonic incubation | Larval rearing | |||
| Parameters | Mean±SD | Range | Mean±SD | Range |
| Water temperature (oC) | 28.11±0.74 | 27-29 | 28.16±0.70 | 27-29 |
| Dissolved oxygen (mg L–1) | 6.43±0.15 | 6.2-6.7 | 6.45±0.12 | 6.2-6.7 |
| Alkalinity (mg L–1 as CaCO3) | 63±1.73 | 60-65 | 63.42±1.63 | 60-66 |
| Hardness (mg L–1 as CaCO3) | 64.36±3.73 | 58-70 | 64.38±3.42 | 58-70 |
| pH | 6.44±0.20 | 6.2-6.7 | 6.43±0.20 | 6.1-6.7 |
| Nitrite-nitrogen (mg L–1) | 0.02±0.007 | 0.01-0.03 | 0.02±0.008 | 0.01-0.03 |
| Table 2: Growth performance and mortality rate of giant gourami for 20 days experiment (SD±) | |||||
| Dosages (mg L–1) | |||||
| Variables | 0 (P1) | 50 (P2) | 100 (P3) | 150 (P4) | 200 (P5) |
| Fertility rate (%) | 89.78±1.68a | 91.11±1.02ab | 92.89±1.02c | 95.11±1.02d | 98.22±0.77e |
| Hatching rate (%) | 78.59±1.57a | 78.59±1.57ab | 84.03±1.939c | 91.35±0.4d | 94.62±2.63e |
| Survival endogenous feeding period (%) | 93.08±1.60a | 95.12±0.90b | 96.18±0.80c | 97.90±0.02d | 99.10±0.39e |
| Weight gain (%) | 828.43±45.65a | 934.07±64.71ab | 985.29±110.92ac | 985.29±181.50d | 1353.19±46.30e |
| SGR (%/day) | 11.14±0.25a | 11.68±0.31ab | 11.92±0.53ac | 12.86±0.69d | 13.38±0.16e |
| Daily weight gain (mg/day) | 5.63±0.3a | 6.35±0.44ab | 6.70±0.75ac | 15.39±1.23d | 19.20±0.31e |
| Thermal growth coefficient (TGC) | 33.99±2.07a | 38.78±2.93ab | 41.10±5.03ac | 51.27±8.23d | 57.78±2.10e |
| Fulton’s condition factor | 1.68±1.57 | 1.70±0.40 | 1.41±0.32 | 1.66±0.60 | 1.49±0.17 |
| Yield (mg L–1) | 199.37±14.36a | 247.29±27.66ab | 265.71±29.56c | 336.38±46.22d | 373.16±5.46e |
| Mortality rate (%) | 21.11±1.92a | 12.22±5.09b | 10.00±3.33c | 5.56±1.92d | 5.56±1.92e |
| Coefficient of variation (CV) of length (%) | 7.36±0.52a | 9.87±0.97b | 6.88±0.47c | 11.50±1.37d | 3.80±0.15e |
| Coefficient of variation (CV) of weight (%) | 4.92±0.25a | 4.92±0.25b | 10.30±1.12c | 14.04±1.94d | 3.19±0.10e |
Data are presented as Mean±SD of three replicates. Different superscripts alphabets in the same row were significantly different (p<0.05) |
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DISCUSSION
The water alkalinity during embryonic development and larval of giant gourami reared ranges from 60-66 mg L–1 as CaCO3. At the same time, pH ranging from 6.1-6.7 and nitrate-nitrogen were between 0.01-0.03 mg L–1. Water quality for the embryonic development and larval nursed system is essential in hatchery operators to enhance the hatching rate, growth rate and decrease larvae mortality6,23,25,26. pH, alkalinity and hardness are water quality parameters critical to water supply and use and larval production during reared in hatcheries23. Aquatic temperature exerts on the metabolic rate of water animals, which results in altered embryonic development and growth in the larvae19. The water temperature during embryonic development and larval reared ranges from 27.8 and 28.2 . According to Prakoso et al.27, the optimum temperature for larva rearing of giant gourami was 30 . Water temperature outside the optimal range (when too high or low) can slow or speed up the incubation time of eggs and cause embryonic death27-29. Water temperature also dramatically influences fish eggs and larvae8. In this study, the dissolved oxygen fluctuated from 6.2 and 6.7 mg L–1 and gave for embryonic support development, endogenous feeding period survival and growth rate of giant gourami. The temperature has supplied sufficient oxygen for larvae survival.
On the other hand, total alkalinity ranged between 60 and 66 mg L–1 as CaCO3 and hardness ranged between 58 and 70 mg L–1 as CaCO3. At the same time, pH ranges were between 6.1 and 6.7. All water quality parameters can support the development of embryonic and larvae of giant gourami. In addition to the water quality parameters mentioned above, others may support Giant gourami embryos and larvae development. These factors are minerals (namely Na, Ca, K, Mg, Fe, Zn, Mn) contained in water with added vitamin C. However, we have not analyzed these elements in this experiment. In contrast, the concentration of ortho-phosphate, nitrate and heavy metal such as arsenic and copper affect the embryonic and larval development of common carp, Cyprinus carpio30. How this parameter itself influences the embryos and larval of giant gourami are poorly understood.
In the present study, egg immersion dosages significantly influenced the giant gourami egg's fertility rate and hatching rate with vitamin C levels except between P1 and P2. It also directly affects the endogenous feeding period survival. The egg immersion with P4 and P5 showed higher fertility, hatching and endogenous feeding period survival than P1, P2 and P3 experiments. These factors may be due to egg immersion with vitamin C dosages, much as 150 and 200 mg L–1, which can contribute to embryonic development, resulting in higher egg hatchability and embryo survival while utilizing endogenous feeding. According to Sulaeman and Fotedar31, at the time of the cycle of endogenous feeding, absorption of nutrients from egg yolk is significant for larvae. Therefore, the method of immersion eggs with vitamins requires high concentrations in incubation water, including vitamin C32. The dosages of vitamin C may also vary during gonad maturation or larva metamorphosis11,21. Thus, giant gourami larvae can increase the tissue levels of vitamin C when fed in sufficient quantities and provide additional protection against environmental stress. Chen et al.33 states that for reducing mortality of golden shiner (Notemigonus crysoleucas) larvae, ascorbic acid can be complete their diet.
The present study recorded weight gain and a specific moderate growth rate in the P2 and P3. In contrast, P1 was recorded lower growth due to the lower dosages of vitamin C in egg incubation water media. This factor might have modulated the physiology and reduce the appetite of the larvae. Ascorbic acid or vitamin C is crucial during ontogenesis because it can minimize larval mortality34. In addition, several authors have linked an ascorbic acid deficiency in fish larvae with collagen and cartilage hyperplasia, scoliosis, lordosis, internal bleeding, opercular reabsorption and abnormal supporting cartilage in the gills, spine and fins with deformation of the jaw and snout35-37.
How dosages of vitamin C itself influence the cartilage hyperplasia, scoliosis, lordosis, internal bleeding, opercular reabsorption and abnormal supporting cartilage of the gills, spine and fins with deformation of the jaw and snout in the giant gourami larvae are poorly understood. However, Furuita et al.20 found a clear relationship between high concentration vitamin C and better eggs quality and increased the survival rate in Japanese eels (Anguilla japonica). Our hypothesis supports that egg immersion of giant gourami with different dosages of vitamin C could improve hatching rate, growth and larval survival. This experiment strengthens vitamin C's role in maintaining normal physiological conditions embryos dan larva of giant gourami. This phenomenon suggests that giant gourami larvae may need high concentrations of water-soluble vitamin C compared to other fish species. For example, immersion rainbow trout larvae with vitamin C did not become essential in increasing their growth38. On the contrary, vitamin C supplementation in the feed played a more critical role in boosting fish growth32. However, the relationship between vitamin concentration and egg quality depends on the type, water-soluble or fat-soluble, or egg quality parameter of the fish19. In this study, vitamin C, a water-soluble vitamin, is needed to develop giant gourami larvae.
CONCLUSION
In conclusion, immersion giant gourami (Osphronemus goramy) eggs and larvae with different doses of vitamin C significantly affected hatchability, survival, growth and yield of larvae. Doses of 150 and 200 mg L–1 are the recommended optimum doses. In subsequent studies, immersion of larvae after 20 days of age with vitamin C is essential to increase survival and growth.
SIGNIFICANCE STATEMENT
Immersion of giant gourami eggs and larvae at a dose of vitamin C of up to 200 mg L–1 is a method to increase hatchability, survival and growth. This study will help the researcher uncover that vitamin C is standardized and widely practised for quality larval production giant gourami. This finding is to overcome the dependability of giant gourami of fish farmers on fry availability is a limiting factor in increasing production in the future.
ACKNOWLEDGMENTS
Authors thank Research Installation for Aquaculture Laboratory Faculty Fisheries and Marine Universitas Riau, which has facilitated this study, fish farmers and students who helped the author during data collection in the field.