The use of Chromolaena odorata leaves to prevent fungal infection on eggs of Pangasius sp.

. There are several obstacles in Pangasius hatchery such as low hatching rate and poor survival. One of the factors that cause low hatching rate on Pangasius sp. eggs is the fungal infection of Saprolegnia sp. Using plant extracts such as Chromolaena odorata leaves can prevent fungal infection because they have antifungal compounds. The objective of the study was to investigate the effect of C. odorata leaves extracts on the hatching rate of Pangasius sp. eggs. This research was conducted in March 2023 at Fish Seed Center, Southwest Aceh Regency. The method used in this study was Completely Randomized Design (CRD) with 5 levels of treatment and 4 replications. The concentration C. odorata leaves extracts used were 0 ppm (control), 100 ppm, 120 ppm, 140 ppm and 160 ppm. Results revealed that the addition of C. odorata leaves extracts have a significant effect (P <0.05) on the hatching rate of Pangasius sp. eggs and the survival of Pangasius sp. larvae. Furthermore, it was found that the best treatment in this study was at concentration of 140 ppm of C. odorata leaves extracts, which resulted in a hatching rate of 76.25% and survival of 90.83%.


Introduction
Pangasius sp. is a freshwater fish commodity that is increasingly demand in Indonesia.This fish is generally found in large rivers in Sumatera, Kalimantan, Java and is a freshwater fish that is cultivated intensively by rearing primarily in ponds [1,2].However, Pangasius hatchery are still facing some problems, one of the obstacles to success in fish farming Pangasius sp. is a disease that attacks both adult fish, seeds, even in the egg phase.There is a disease attack caused by fungus during egg hatching has an impact on egg damage that causes a decrease in hatchability [3,4].Moreover, [5] mentioned that low hatching rate is a problem that often occurs in Pangasius hatcheries.Additionally, previous research emphasized that the fungus that attacked fish eggs in cultivation activities was Saprolegnia sp. which can reduce hatching rates [6].Saprolegnia is one of the most common aquatic diseases in freshwater fish and affects all stages of the fish life cycle [7,8].The characteristics of fish eggs attacked by the Saprolegnia fungus are that the fish eggs grow white threads like cotton on the surface of the fish eggs, so that the eggs rot and do not hatch [9,10].Furthermore, eggs that have been infected with the fungus Saprolegnia sp.there will be changes in morphology or growth of fungus on the egg, marked by the growth of fine threads resembling cotton on the surface of the egg in the form of white fibers or what are usually called hyphae [11,12].
Prevention of fungal attacks on eggs has been tried using chemicals.However, the risk of using chemicals or antibiotics can cause water pollution, because these chemicals are difficult to decompose and they can have dangerous effects on the organism that use them and the environment itself [11,13].Therefore, natural ingredients are used that have anti-fungal properties.Besides being cheap, natural materials are also safe for the aquatic environment because they decompose easily.One way to control Saprolegnia fungus attacks is to use plant extract that have antifungal properties.Moreover, various types of plant extract have been studied to prevent fungal infections on egg stage such as Cassia alata L. [4], tea leaf [3], Allium sativum [14][15][16], Piper betle L. [11,17] and other plants that can be used as an antifungal agent in fish eggs namely Chromolaena odorata.C. odorata are a type of plant from the Asteraceae family.According to phytochemical test, C. odorata leaves contain several main compounds such as tannins, phenolics, flavonoids, saponins, steroids, alkaloids, flavanone, essential oils and terpenoids [18][19][20][21][22]. Previous works on C. odorata has been carried out particularly on other species such as Chanos chanos [23], Clarias gariepinus [24].There is no available information currently about the use of C. odorata leaves extracts for fungal prevention on Pangasius eggs.Therefore, the objective of this study was to investigate the effect of C. odorata leaves extracts on the hatching rate and survival of Pangasius eggs.

Experimental design
This research was conducted at Fish Seed Center, Southwest Aceh Regency in March 2023, using experimental methods which designed as completely randomized design (CRD) consisting of 5 treatment levels and 4 repetitions.The treatments tested in this study were different concentration of C. odorata leaves extract, namely treatment A (0 ppm), B (100 (ppm), C (120 ppm), D (140 ppm), and E (160 ppm).

Chromolaena odorata leaves extract preparation
Extracts of C. odorata leaves was made based on study by [23] which used maseration method.The C. odorata leaves washed and dried, then blended until they became powder, and weighed as a dry weight of 300 grams of flour.C. odorata leaves which have become flour were soaked in 96% ethanol for 24 hours and evaporated using a rotary evaporator until they became a filtrate.The filtrate was then stored in the refrigerator.

Eggs Immersion
The eggs used for this research were obtained from the artificial spawning of Pangasius sp.Pangasius sp. were reared based on the guidelines of the Institutional Animal Care and Use Committee (IACUC, 2018) and have ethical approval B/034SN/XII/2022 from the animal ethic committee of Marine and Fisheries Faculty, Universitas Syiah Kuala.The number of eggs used in this research was 2000 eggs, each experimental unit consisted of 100 healthy fertilized eggs.Fertilized eggs are characterized by the presence of a developing nucleus and a transparent color.Meanwhile, unfertilized eggs are white color [25,14].The eggs were transferred to each soaking container which had been given C. odorata leaves extract according to the specified dose.This soaking was carried out for 30 minutes for each treatment [23].Next, the eggs are removed and transferred to the hatching container.Then the development of the eggs was measured using a microscope.The embryogenesis process of egg development was observed once every hour.

Hatching Rate (HR)
Hatching Rate (HR) were calculated by following formula [26]: Total number of eggs hatched Total fertilized eggs x100 (1)

Larval Survival Rate (SR)
Larval survival (SR) can be determined at the end of the study.Observations of larval survival were carried out for 10 days from the start of larval rearing.The survival rate of larvae can be calculated using the [27] formula as follows: Where Nt is number of larvae at the end of study and No is initial number of larvae.

Water Quality
The water quality observed was temperature, pH, and DO.Water quality was measured in the morning and evening.

Statistical Analysis
Collected data were analysed with statistical software using one-way analysis of variance (ANOVA).Then, the further test, least significant difference (LSD) was applied when statistical differences were observed (p < 0.05).Embryonic development of Pangasius was explained descriptively.

Embryonic Development
The results of the development of Pangasius sp.eggs soaked using C. odorata extract can be seen in Table 1.
, The formation that occurs after the fertilization phase, then the egg cell undergoes division.

Water Quality
The results of water quality measurements during the research included measurements of temperature, pH and DO.During research, it showed that the water quality was still relatively good.

Embryonic development
The development egg stages into a larva starts with the cell division (cleavage) phase of the morula, blastula, gastrula, organogenesis and the egg hatches into a larva.Pangasius eggs were observed under a microscope with a magnification of 10x40µ, it showed that the development of embryo had entered the morula phase, because the cell division phase had passed.According to [28], the cleavage phase is characterized by blastocyst formation at the animal pole.Complete blastocyst formation occurs 60 minutes after fertilization.Then this blastocyst will divide to form 2, 4, 8, 16 and 32 cells within 55 minutes, and will enter the morula phase after dividing into 32 cells.Basically, cleavage is a process of cell division during embryonic development, the size of which becomes smaller and smaller over time or becomes small units called blastomeres [29].
The morula is the phase where the blastomeres that are formed will condense at the animal pole to form two cell layers [30].According to [31] the morula stage is the end of the cleavage phase which will then be continued with the stage of embryonic organ formation.Another opinion stated that the morula phase ends when blastomeres are produced.The blastomeres then condense into small blastodes forming two cell layers and at the end of division two groups will be produced [32].In this study, the morula phase lasted 3 hours.In the research of [33], this phase lasts 2 hours.
The Pangasius embryo continues to undergo cell division until it enters the blastula phase.In this phase the blastomeres divide into smaller ones, so that in the morula stage the blastomeres which were originally solid will form an empty space called the blastosule [30].[34] stated that the blastomeres moved in both the side of animal pole covering 30-40% area over the yolk sphere.The blastula phase in this study occurred for 3 hours.This is in line with research by [33] which states that this phase lasts 3 hours.
The next phase is the gastrula, according to [30] who said that the gastrula phase is characterized by the process of expanding and closing the egg yolk by the blastoderma towards the blastopore, so that the entire yolk is covered by the blastoderma.[35] stated that the gastrula phase is characterized by the blastoderma covering almost the entire egg yolk, the part of the egg yolk that is not covered is called the blastopore.In this phase, you can see the rotating movement of the cells, the organs of the catfish begin to form in this phase.This phase lasts 4 hours.In the research of [33], this phase lasts 3 hours.
Organogenesis is characterized by the formation of a head and tail.The results of observations in this phase show that there is movement of the embryo.In this study, the organogenesis phase occurred for 9 hours.In the research of [33], this phase occurred lasts 8 hours.The occurrence of the organogenesis phase according to [30] is characterized by the formation of the head and tail, spine segments, eyes, heart, egg yolk pigmentation and hatching into larvae.
The final phase, namely egg hatching, is marked by the embryo actively moving around to change its position, this is because the space for movement is smaller compared to the growing size of the embryo, this phase lasts 4 hours.This is in accordance with what was stated by [36] that when an egg is about to hatch, the embryo often changes its position due to lack of space in its shell.In the research of [33], egg hatching occurred lasts 2 hours.In this study, Pangasius eggs hatched for 24 hours, this is in accordance with the statement of [37] which stated that the hatching time for catfish eggs ranged from 20-26 hours.[38] also revealed that eggs hatching of Pangasionodon hypopthalmus took 24 hours.However, the length of time for egg hatching in this study is different from the research of [33], namely for 25 hours.According to [39], the newly hatched larvae of Thai Pangasius are quite transparent , 03017 (2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301787 and light yellowish in color with a body length of 2.98-3.10mm.[40] mentioned that newly hatched larvae of Pangasius hypophthalmus ranged from 2.74-3.00mm total length.

Hatching rate and larval survival of Pangasius sp.
Based on Analysis of Variance (ANOVA) test, use of C. odorata leaves extract have a significant effect (P<0.05) on hatching rate of eggs and larval survival of Pangasius sp.The further test of Least Significant Different (LSD) revealed that treatment D (140 ppm extract of C. odorata leaves) had the highest egg hatchability and larval survival of Pangasius sp. with values of 76,25% and 90,83% respectively.It was assumed that 140 ppm extract was the optimum concentration for inhibiting growth of fungus on eggs and larvae.Furthermore, this statement is in accordance with [4,15] that the use of anti-fungal agents at normal concentrations inhibits fungal growth, but at higher concentrations it can damage egg cell tissue.Another statement was revealed by [41] which reported that C. odorata leaves contain alkaloids, flavonoids, tannins and saponins as antifungals in fish eggs.Fungistatic antimicrobial substances inhibit the work of certain enzymes which result in disruption of fungal cell metabolism, so that the process of elongation of fungal hyphae (mycelium) is hampered.If the growth of fungal cells, which is characterized by the elongation of hyphae (mycellia), is hampered, the fragmentation of the hyphae becomes disturbed, so it can be said that the fungal cells cannot reproduce.Hyphae or mycelium which cannot experience fragmentation are caused by damage to the hyphal tissue, so that the fungal cells die easily [4].This showed that C. odorata leave extract is able to inhibit the growth of fungus in Pangasius eggs.
However, at the highest concentration, treatment E (160 ppm), showed that the egg hatchability value decreased and this was followed by a decrease in larval survival.This is thought to be because the extract concentration is too high, causing the fish eggs to not hatch.This is in line with research by [16], namely the use of garlic extracts on the hatchability of milkfish eggs experienced a decrease in the percentage of egg hatchability (63.66%) at high concentrations (2.500 ppm).Research by [17] explained that the use of Piper betle L extract at a high concentration, namely 1.2 g/L, caused a decrease in the hatchability of catfish eggs (50.88%), the higher the concentration of Piper betle leaf extract will not only kill the fungus but will also be absorbed by the egg within an intolerable limit so that it is toxic to the egg and causes the egg to not hatch.Moreover, the use of antifungal agents at normal concentrations inhibits the growth of fungi, but at higher concentrations it can damage the chorion layer on the egg, causing the egg to shrink so that it cannot hatch.Apart from that, active compounds such as saponins, which are anti-microbial compounds, can be toxic to organisms at high concentrations.Saponin compounds are also widely used as fish poison [42,43].
According to the previous study on other species, found that the best concentration of C. odorata leave extract was at 120 ppm on Chanos chanos with 85% of hatching rate [23], whereas [24] reported that the highest hatching rate of Clarias gariepinus was achieved at 200 ppm C. odorata leave extract with 80.66% of hatching rate.The difference in optimum concentration obtained on different species.
As shown in Table 2, treatment A as a control without used C. odorata leave extract had the lowest egg hatchability, namely 41.50%.It was observed that eggs were attacked by fungus.This is in line with [4,14] statement, which stated that eggs that were not soaked with antifungal agents accelerated the attack of the Saprolegnia fungus and covered the chorion, Saprolegnia sp. can weaken the stiffness of the chorion so that it wrinkles and then dies, causing the eggs could not hatch.A previous study of [44] described that other factors that can cause low levels of egg hatching are eggs not developing after being fertilized, changes in the physiological abilities of eggs during embryogenesis.Another statement by , 03017 (2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301787 [45] mentioned that not all fertilized eggs will hatch into larvae.Eggs that do not hatch can also be caused by poor condition of the eggs due to the presence of a mixture of water when taking the eggs.
The percentage survival value of catfish larvae in this study was 90.83%.The results in this study were classified as good for all treatments.This statement is in accordance with [46], which stated that the average survival rate for catfish ranged between 70 and 80%.The survival results in this study were not much different from those obtained by [47], namely the percentage of catfish survival obtained was 97.31%.Meanwhile, in research [3], the highest survival was obtained at 89,27%.According to [48] the survival rate of fish is influenced by two factors, namely internal factors and external factors.Internal factors that influence are competitors, parasites, population density, adaptability, and human handling.External factors that influence are the physicochemical properties of waters.

Water Quality
Water quality parameters are important in the process of hatching eggs and rearing larvae.This is in accordance with the statement of [49] who said that water quality parameters are external factors that influence fish reproduction and egg hatching.The water quality parameters observed during the research were temperature, pH and dissolved oxygen (DO).The average temperature values obtained during the research were 26-28C, pH 6.8-7.5 and DO 5-6 mg/l (Table 3).The parameter values obtained are in accordance with the statement by [50], who said that in order for catfish eggs to hatch well, the eggs must always be submerged in water and the water temperature must be kept stable in the range of 24-30°C.If the temperature is too low, egg hatching will take longer, while if it is too high, the egg will not hatch.In this study, egg hatching occurred within 24 hours.The pH value obtained in this study is in accordance with [51] statement which states that a good pH for hatching fish eggs ranges from 6.5-8.DO value in this study is in accordance with the statement by [52] which says that dissolved oxygen is good for hatching fish eggs, namely >5 mg/l.

Conclusion
Use of Chromolaena odorata leaves extract affects the hatchability and larval survival of Pangasius sp.The highest percentage of egg hatchability and larval survival were obtained at 140 ppm extract, namely 76.25% and 90.83%, respectively. /doi.org/10.1051/bioconf/2024870301787 2024) BIO Web of Conferences ICFAES 2023 https://doi.org/10.1051/bioconf/2024870301787 Eggs hatching 21st-24th hour (4 am-7 am) The process of movement of the embryo out of its shell 3.2 Hatching rate of eggs and Survival of Larvae of Pangasius sp.Use of C. odorata leaves extract on this study resulted the hatching rate of eggs ranged from 41.50 ± 3.41% to 76.25 ± 3.86% and survival of larvae ranging from 78.54 ± 6.58% to 90.83 ± 1.68%.

Table 1 .
Observation of embryonic development

Table 2 .
Hatching rate and Larval survival a Note : The average values in the same column with different superscripts indicate that they are significantly different and the values with the same superscript in the column indicate that they are not significantly different.