Effects of different substrates and feeds on worm biomass (Limnodrilus hoffmeisteri)

Trinh Thi Lan^1,2, Nguyen Thi Bich Hanh^1,2, Nguyen Thi Thuy Hang^1,2, Nguyen Thanh Sang³, Do Dai Phat³, Nguyen Trung Duong³ and Nguyen Huu Yen Nhi^1,2

¹ Department of Aquaculture, Faculty of Agriculture and Natural Resources, An Giang university, Vietnam
nhynhi@agu.edu.vn
² Viet Nam National University, Ho Chi Minh City,Vietnam
³ Student of Aquaculture Department, Faculty of Agriculture and Natural Resources, An Giang university, Vietnam

Abstract

The objective of the study was to find the optimal substrate and feed for the biomass of the worm. The experiment was arranged in a completely randomized design including 2 factors (substrate and feed) with 9 treatments and 3 replications. Three types of substrates (CN) were CN1 (2kg of catfish pond mud + 0.5kg of duckweed); CN2 (1kg of catfish pond mud + 1kg of chicken manure + 0.5kg of duckweed); CN3 (2kg of chicken manure + 0.5kg of duckweed). The three types of feed used were compound feed for fish fry (35% Nitrogen), rice bran and cow manure. The environmental factors throughout the experiment were maintained stable throughout the rearing process to ensure the development of the worms. The temperature was stable at around 27^oC, the pH in the rearing tray was stable at 6.96, and the dissolved oxygen was maintained at around 4.3mg/L. In particular, the water flow rate did not exceed 250ml/min. The experimental results showed that the treatment combined CN1 (2kg of catfish pond mud + 0.5kg of duckweed) as a substrate and supplemented with compound feed gave the best results in terms of biomass and density of the worms and was statistically significantly different from the other treatments. The treatments using catfish pond mud plus duckweed as a substrate as well as feeding with compound feed had the highest biomass and density compare with the rest groups. The protein composition in worm was highest in compound feed as well as chicken manure plus duckweed substrate groups and was significant different with other groups. Thus, the process of cultivating the worms biomass in the recirculating system was suitable in catfish pond mud plus duckweed as well as feeding compound feed.

Keywords: fish fry, substrate, catfish pond mud, supplementary feed, worms, Tubifex

Introduction

Limnodrilus hoffmeisteri worms, also known as silkworms or red worms or bloodworms. The use of tubificid worms (Tubifex sp. or Limnodrilus sp.) as live feed for fish during the transition from hatchlings to juvenile stages has been shown to be critically important for ensuring rapid growth, high survival rates, and physiological development. Simangunsong et al (2024) emphasized that tubificid worms are a natural feed rich in protein, unsaturated fatty acids, and easily digestible nutrients, making them ideal for larval fish with developing digestive systems. Syamsuddin and Rachmawati (2021) confirmed that supplementing diets with Tubifex significantly improves growth, pigmentation, and resistance in angelfish. Similarly, Sunarno et al (2023) noted enhanced feeding stimulation and feed conversion efficiency in fish fed with tubificid worms.

However, harvesting Tubifex from natural environments (such as canals or sludge-rich pond bottoms) poses a significant risk of introducing pathogens (bacteria, viruses, parasites), which threatens biosecurity in hatcheries. Thus, studies highlight the importance of culturing tubificid worms in control, biosecure systems by treating the substrate to culture worms that can serve as a safer protein source for Pangasius fry, reducing gastrointestinal disease risks.

Tubificid worms have been identified as a high-protein, high-amino-acid-profile source for fish growth (Alam et al 2021). This worm has a large market in fish hatcheries, particularly in spawn production of catfishes and rearing of ornamental fishes (Mandal et al 2016). In Vietnam and Bangladesh, for instance, the existing supply of tubificid worms comes from wild sources that are inconsistent and unreliable in meeting hatchery demand. Besides, in the wastewater discharge from Pangasius pond that create the scarcity of tubificid worm’s collection. Moreover, collection from sludge environments lacks purity and poses a risk of pathogen transmission to fish (Mandal et al 2016). Therefore, the culture of Tubifex in captivity is necessary to ensure consistent supply and safe feed input. Meanwhile, the global demand for ornamental fish is increasing and is now considered a commercial industry due to its profitability (Ansari et al 2014). The growth and survival of pangasius fish and ornamental fish are highly influenced by the type of feed ingested (Andriani et al 2020). Sludge worms are among the most important live feeds, serving as essential nutritional sources of carbohydrates, lipids, and proteins (Conceição et al 2010). High-protein feed is required for ornamental fish farming but can elevate production costs (Mente et al 2017). Thus, cultured worms offers a cost-effective, nutritious solution, control biosecurity for enhancing larval performance and reducing risks.

Some authors such as Marian and Pandian (1984) and Marian et al (1989) have tried to develop this species and have achieved some significant successes. Oplinger et al (2011) studied the initial stocking density affecting the growth and biomass productivity of worms when reared at 7 density levels of 2,675 - 267,451 individuals/m² for 120 days. The results showed that the initial stocking density of 2,675 individuals/m² gave the highest biomass. In addition, Nhan (1999) studied the morphological characteristics and distribution of the worm (Limnodrilus hoffmeisteri) in freshwater aquaculture ponds and found that the average length of the collected worm individuals was 21.34 ± 5.76 mm. In addition, Hong et al (2014) studied the growth and density increase of the worm population on different food sources such as rice bran, chicken manure and cow manure and found that the worm biomass as well as the density of the worm individuals increased the highest when fed with rice bran and the lowest in the treatment of feeding with chicken manure and feeding with cow manure. In addition, Trang et al (2019) also studied the effects of different types of substrates on the growth and biomass of worms under artificial conditions, showing that the substrate of 50% catfish pond mud + 50% chicken manure gave the highest density of worms at 87,125 ± 14,766 individuals/m². Furthermore, Mahendra et al (2019) studied the effects of using duckweed and mud as substrates on the biomass of worms also showing that the combination of 100% duckweed + 2000g of mud gave the best results with a density of 6,250 individuals/gram and a biomass of 8.60 mg/cm². From the previous studies summarized above, it can be seen that each author used different types of food or different substrates. No author has yet synthesized the types of food and substrate to come up with a suitable farming process under Vietnamese conditions, thus this study was established to find the optimal substrate and feed for the biomass of the worm.

Materials and methods

Experimetal location

The experiment was conducted in experimental farm of Aquaculture Department, Faculty of Agriculture and Nature resources, Angiang University, An Giang province, Vietnam.

Substrate, feed and seed worms

- Catfish pond mud: Mud from bottom of Pangasius ponds is collected from the farming areas of Pangasius farms. After being transported to the farm, the catfish pond mud was be dried, ground, and worm cocoons and pathogens removed. Then, the mud was packed in plastic bags and stored in a dry place until use.

- Chicken manure: Chicken manure was purchased from reputable farms and processed to increase efficiency. After being brought to the farm, the manure is dried and mixed with secondary EM from the An Giang Provincial Center for Application of Science and Technology Advances, which helps decompose organic compounds, remove NH₃, NO₂, H₂S, and deodorize. The secondary EM is diluted with water (20-30 ml/8 liters of water) and left for 1 hour, then sprayed on the chicken manure. After that, the chicken manure was covered in a thick layer of tarpaulin, creating an anaerobic environment for 1-2 weeks until the manure no longer smells bad. Then, the chicken manure can be bagged until use.

- Cow manure: Cow manure (Photo 4.) was collected from reputable livestock facilities, ensuring quality and source. After that, the cow manure was be treated by composting with Trichoderma microbiological preparation with a dosage of 1kg Trichoderma + 100L of water, soaked for 1-2 days, then sprayed on 100kg of cow manure, helping to decompose organic components in the manure, remove odors, and kill larvae and potentially harmful bacteria. The manure was be composted for 1 to 2 months. During this process, the manure was be checked and mixed every week to ensure even distribution of microorganisms. If the manure was not evenly soaked with microorganisms, Trichoderma can be added with a dosage of 500g Trichoderma + 50L of water and watered evenly on the manure. When the cow manure has completely lost its odor and meets the requirements for decomposition, the straw and grass mixed in the cow manure was be removed. Then dry, put in plastic bag, tie tightly until use.

- Duckweed: Duckweed was collected from areas such as swamps, ponds, and ditches - places where duckweed grows densely. After being brought experimental farm, the duckweed was washed, dried, ground and then packaged for storage

- Rice bran and compound feed were bought form a local market in Long Xuyen city. Compound feed was the commercial feed for striped catfish fingerling with 35% of crude protein, which was ground to feed for worms.

- Worm seed: The worms were purchased directly from people who collected them from Tra fish ponds in Long Xuyen city. After that, the worms were domesticated in tanks to get used to the experimental environment before setting up the experiment.

The chemical composition of seed worms, substrate and feed using in the experiment was showed in Table 1.

Experimental system

The experimental system consists of 27 trays 65x42x16 cm were designed to circulate, connected together by PVC pipes Ø27, the outlet is connected to the mechanical filter tank by a pipe with a diameter of Ø34. The trays were placed on shelves (the tray shelves must be higher than the biological and mechanical filter tanks, so that water can automatically overflow into the mechanical filter tank). At the bottom of the mechanical tank, an open pipe was filled to lead the overflow water to the biological filter tank. Inside the biological filter tank were kanet granules and an oxygen system was installed to provide dissolved oxygen to the water and provide oxygen for the process of decomposing toxic gases. Inside the biological filter tank was installed a 120W submersible pump to lead water from the biological filter tank to the water tank ready to supply to the breeding trays (the ready water tank also helps to adjust the water flow rate from the pump). The ready water tank was placed taller than the trays (so that water can be automatically led from the tank to the trays). The ready water tank was made of a 70 liter plastic barrel. The barrel is punched with a hole from the bottom of the tank to stick a Ø21 PVC pipe to led water to the trays, each outlet of 01 tray was installed with a water valve to adjust the water flow. Above the mouth of the barrel was installed an overflow pipe and brings the extra water back to the biological filter tank.

Experimental treatments and design

The experiment was arranged in a completely randomized design including 2 factors (substrate and feed) with 9 treatments and 3 replications. Three types of substrates (CN) were CN1 (2kg of catfish pond mud + 0.5kg of duckweed); CN2 (1kg of catfish pond mud + 1kg of chicken manure + 0.5kg of duckweed); CN3 (2kg of chicken manure + 0.5kg of duckweed). The three types of feed used were (CF) compound feed for fish fry (35% Nitrogen), (RB) rice bran and (CM) cow manure. Individual treatments were:

Treatment 1: CN1 + CF

Traatment 2: CN2 + CF

Treatment 3: CN3 + CF

Treatment 4: CN1 + RB

Treatment 5: CN2 + RB

Treatment 6: CN3 + RB

Treatmet 7: CN1 + CM

Treatment 8: CN2 + CM

Treatment 9: CN3 + CM

Each worm tray was provided with 4 cm of substrate from the bottom of the tray according to each corresponding treatment (Fig 8 and 9). After arranging the substrate in the trays, the circulating water system was be activated by letting water flow through PVC pipes Ø 21. The water flow in each tray was adjusted through valves, ensuring a flow rate of 250ml/min for each tray. The initial mass of worm seeds released is 10 grams/tray with an initial density of 1.5 worms/cm². The experiment was implemented in 90 days.

Experimetal management

Worms were fed periodically every 2-3 days, depending on the requirements of the worms. Before feeding, the feed was soaked in clean water (enough water to soften the food) for 1 day to decompose the food. It is best to feed worms in the evening (5-7pm). During feeding, turn off the water valve until the feed settles to the bottom, then turn the water back on. Observe the feed in the tray the next morning to adjust the amount of feed accordingly for the next feedings.

Harvesting worms

Referring to the researchs of previous authors, it was recommended to harvest 50% of the worms after 30 days of rearing and continue to add substrate and feed for the rearing process to be effective and continuous (Nhung et al 2016). The worms and substrate were collected and placed separately in different trays. Use a small plastic basin to hold the worms and the collected substrate, then cover it in the dark for about 15 minutes. In the absence of oxygen and dark, the worms was gather on the surface of the substrate, then they were collected in a tray of clean water with aeration to let the water overflow to remove all organic debris. After that, the worms were weighed and counted to calculate the results achieved.

Measurements

- Environmental factors: Temperature was be monitored directly at the trays for timely adjustment. Temperature was stable in the optimal range of 25-28^oC. Other factors such as pH, DO, NH ₃ and NO₂ are twice measured a day using the Sera test.

- Worm biomass: the worms were be weighed before conduct the experiment and after harvesting.

- Count the number of worm per cm²: Count the worm density under a stereo microscope to know the number of worms that have increased during the rearing process.

Statistical analysis

The data were analysed by use a Minitab program with general linear model (GLM) option (version 16.0) ANOVA software Minitab (2010).

Results and discussion

Water quality parameters during the experiment

The environmental factors of experiment in the process of raising worms was showed in Table 2.

The experiment was arranged indoors and in the same system, so the average temperature between the trays and the filter tank did not differ, fluctuating between 27.2 - 27.5^oC and was completely suitable for the growth of the worms. According to Aston (1968), the best temperature for the development of the worms was between 25 - 30^o C, which shows that the temperature during the experiment was completely suitable for the development of the worms.

According to Davis (1974), the worms adapt well to a pH range of 6.0 - 8.0. The pH in the trays was 6.96, in the mechanical filter tank it was 7.08 and in the biological filter tank it was 8.06. The pH was different in the trays and the water treatment tanks, but in general, the pH fluctuated little, not too high nor too low, the pH was within the suitable range for the development of the worms. Low pH in mechanical filter tanks is due to the excess substances in the rearing trays being concentrated in this tank for treatment, so the pH is often low. Then the water is passed through a biological filtration system (with the presence of microorganisms to clean the water), so the pH increases.

Oxygen measurement results show that the average dissolved oxygen content was 4.3 ± 0.32 mg/L in the rearing trays, while in the mechanical and biological filter tanks it was 4.12 ± 0.9 mg/L and 6.98 ± 0.89 mg/L, respectively. According to Marian and Pandian (1984), maintaining an oxygen content of 3 mg/L or higher can increase the density and ensure high reproduction of worms. If the oxygen content was low or less than 2 mg/L, it was inhibit their activity and reproduction, the minimum oxygen content to ensure the survival of worms was 1.7 mg/L. Therefore, the dissolved oxygen content in the experiment not only created conditions for good growth of the worms but also ensured their good reproductive ability.

The factors NH₄⁺/NH₃ and NO₂ ^- during the experiment did not fluctuate much, still within the appropriate range for worm develop well. According to Shafruddin and Efiyanti (2005), ammonia up to 0.28-1.50 mg/L was still good for the growth of worms. Inaddition, Nijboer et al (2004) indicated that worms often have high adaptability in polluted water areas rich in organic compounds.

From these enviromental factors in this experiment were always within the appropriate range for worm development, without affecting the experimental results.

Effects of substrate and feed type on biomass of worms

According to previous research authors such as Nhung et al (2016), Hong et al (2014), Lobo and Alves (2011), Trang et al (2019) and Mahendra et al (2019) the type of substrate and supplementary feed have a great influence on the development of worms.

The results of the effects of substrates and supplementary feed on the weight of worms were shown in Table 3.

Table 3 and Figure 1. showed that when raising worms with different substrates, there was a clear effect on the increase in worm biomass (p1<0.05), which was also consistent with the studies of previous authors. The substrate type of CN1 (2kg of catfish pond mud + 0.5kg of duckweed) grow best, the mass of worms obtained was the highest. The lowest was the substrate of CN2 (1kg of catfish pond mud + 1kg of chicken manure + 0.5kg of duckweed). Similarly, the types of feed also affect the increase in worm mass, the best was supplemented with compound feed, the lowest was fed with cow manure and significant difference between groups (p2<0.05). This result was similar to the results of the study of Hong et al (2014) and Reynoldson et al (1996) when feeding worms with compound feed for ornamental fish and rice bran for quite good worm biomass. The interaction analysis between substrate and supplementary feed on the mass of worms also showed that there was a statistically significant difference (p3< 0.05). The treatment with substrate of CN1 (2kg of catfish pond mud + 0.5kg of duckweed and supplemented with compound feed gave the best and most stable biomass, followed by the combination of substrate of CN1 (2kg of catfish pond mud + 0.5kg of duckweed) with supplemented rice bran, the lowest was substrate of CN3 (2kg of chicken manure + 0.5kg of duckweed) and supplemented with cow manure.

Figure 1. Effects of substrate and feed on final weight of worms (g)

The increasing in biomass of worms was not only reflected in the total mass of worms but also in the worms biomass shown in Table 4.

After 90 days of rearing. the best worm biomass was achieved in the treatments using substrate CN1 (2kg of catfish pond mud + 0.5kg of duckweed) with average was 0.603 g/cm². The lowest was in the substrate CN2 (1kg of catfish pond mud + 1kg of chicken manure + 0.5kg of duckweed) was 0.41 g/cm². This difference was very clear and statistically significant (p2<0.05). The worm biomass was also highest in the treatments supplemented with compound feed (0.785 g/cm²) and was significant different from the treatments fed with the other feed. The influence of substrate and feed types on worm biomass was very clear, the most clearly in the treatment combining CN1 and compound feed and significant difference with the rest treatments (P3<0.05).

The increase in biomass of worms was also shown by increasing the density of worms. The results in Table 5 show the effects of substrate and feed on the density of worms in the experiment.

The density of the worms was greatly affected by the types of substrates and supplementary feeds. which was consistent with the observations of the authors Lobo and Alves (2011), Hong et al (2014), Trang et al (2019) and Nhung et al (2016). The difference in the combined effects of different types of substrates and feeds between the experiments has a significant difference in the density of the worms obtained (p3< 0.05).

To evaluate the effectiveness of raising a certain object, the feed conversion ratio (FCR) index shows this. The results of the FCR of the worms are shown in Table 6.

The best FCR was in the treatments feeding compound feed or rice bran combined with substrate CN1 (2kg of catfish pond mud + 0.5kg of duckweed). The highest FCR was in the treatment using substrate CN3 (2kg of chicken manure + 0.5kg of duckweed) combined with feeding cow manure. These results was also consistent with the results of previous authors that feeding cow manure did not increase the biomass of worms (Oplinger et al 2011). Inaddition, feeding chicken manure as substrate, the efficiency was not as high as when combined with mud or clay as well as fine sand.

Although the biomass results in this experiment feeding with cow manure and chicken manure as substrate were not as high as the remaining experiments, but they were still higher than the results of previous authors. The reason for beter result maybe due to the addition of a certain amount of duckweed into the substrate of all treatments, which increase the organic matter content to help the worms grow well.

The protein content of the worms obtained after the experiment was showed in the Table 7

Table 6 showed that substrate and supplementary feed have direct effects on the protein content of worms. For substrate, the best protein content in worms was obtained in substrate CN3 (2kg of chicken manure + 0.5kg of duckweed) with average protein content in worms was 49.87%. In term of supplementary feed, the highest protein content of worms using compound feed was 50.35%. Analyzing the interaction between substrate and feed, the treatments using CN3 or feeding compound feed gave the best result and no significant difference between these treatments.

Conclusion

The combination of catfish pond mud with duckweed as substrate and supplemented with compound feed gave the best results in biomass and density of worms and was statistically different from the other treatments.

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