Showing posts with label rptr. Show all posts
Showing posts with label rptr. Show all posts

Research Paper to Read: (2014) The Study of Different Foods on Spawning Efficiency of Siamese Fighting Fish (Species: Betta splendens, Family: Belontiidae)

Source: ResearchGate.

What are the key point here?
(1) There is no significant differences between live feeds (Artemia, Blood worm, and Gammarus) and artificial feeds in Betta sp. breeding.

Why this paper?
As fish diet is a crucial nourishment for the brood stock in conditioning Betta sp. for breeding. What are the possible best food available to obtain a large spawn? Having a large spawn increases the breeder farm profits as the farm owner sell more fishes. This question have been asked numerous time in online forums and the general consensus among the breeders is use live foods with high protein nutrients. However, obtaining and culturing live feeds do incur additional costs and labour. Thus, through this paper, we would like to know what are the significant differences in using different foods and if those differences worth using it. (Note: This is not a the best written paper but we are always curious about the materials and methods used in breeding. Either it reconfirm our understanding or raises further questions regarding our current approach.)

Material and Methods
This experiment was conducted in a commercial fish farm which produces 50000 fishes annually. The material used are 20 aquariums, 40 fishes, 5 different fish feeds with 4 replicates as shown below. The dimension of each aquarium is 40cm x 30cm x 30cm with water height around 15cm, which is 18 litres. (Note: Not sure about the 'mass', assumed it refers to a group of egg.)

(1) Artermia cyst x 4 pairs.
(2) Artermia mass x 4 pairs.
(3) Blood worm x 4 pairs.
(4) Gammarus mass x 4 pairs.
(5) Commercial food x 4 pairs.

The water parameters of the breeding tank as below:

(1) pH: 7.5.
(2) Air temperature: 30 - 35 Celsius.
(3) Water temperature: 26 - 28 Celsius.
(4) Hardness: 108 ppm.
(5) Dissolved oxygen: 6 mg / litre.

The breeding procedures as follows:

(1) The breeding tanks were disinfected with salt without iodine.
(2) Fishes were disinfected using solution of 5% Acrofelavin bath for 5 minutes.
(3) Once the brood stocks were added to the breeding tank, it's not feed for 24 hours.
(4) A glass screen was added between the pair of fishes.
(5) Fishes were fed twice (9 a.m. and 6 p.m.) on daily basis on ratio of 3% of body weight of the fish.
(6) Thick plastic with measurement of 10 cm x 10 cm was added to the side of male fish.
(7) The pair of fish was conditioned for 15 days.
(8) Female fish was released to the male once the bubble nest size reaches 500 bubbles.
(9) Once spawning is done, the female is extracted out from the breeding tank.

During the incubation period, the temperature of the breeding tank is controlled with a digital thermometer (VIPRO) with a 0.1 Celsius minuteness. The temperature is set at 27 Celsius. 10 eggs were randomly selected from each breeding tank and were measured using a digital micrometer (Leitz, model 621).

From the table below, it's shown that there is no significant differences (P >= 0.05) between different food treatments even though the pair fed with blood worms produced the most eggs and the lowest spawning time. (Note: Table below is not the best way to present your tabulated data).

While the feeds doesn't show any significant impact in affecting the eggs production. As the brood stock is fed twice per days, if the feeding frequency increased to 4 times / day, will it impact the result? While live feed like Blood worms are rich with protein, does artificial feeds with similar protein percentage may have the similar or better results?

What you're going to do with the knowledge you've gained?
(1) Conditioning for 14 days or 2 weeks is crucial to make sure the pair of brood stock breed. Thus, we will need get a good partition to separate both fishes instead of using plastic container to hold the female Betta sp.

(2) Where can we get Gammarus?

What are the further unsolved questions?
(1) What is the optimum tank size suitable for breeding Betta sp.?
(2) What is the optimum feeding frequency suitable for the brood stock?

Note to Self:
(1) Be careful when selecting papers to read. Always check the references section to determine whether this is a significant paper to read.

(2) Paper with plenty of grammatical and spelling errors is a good indicator to skip it.

Research Paper to Read: (2009) The Siamese fighting fish: Well-known generally but little-known scientifically

Source: Research Gate.

What are the key point here?
(1) There are 55 species of Bettas in Indochina and can be grouped into either nest building or mouth brooding care. Thailand have 10 wild Bettas where 4 are bubble-nest builders and 6 are mouth brooders. (Note: I'm quite surprised and thought TH have more, it seemed MY have even more wild Betta sp.)

(2) More research should be done locally but instead more publications are published by those living overseas with where Betta sp. is not abundance. (Note: Something similar to MY where most publications on Betta sp. came from SG researcher)

Why this paper?
As the common name implies, Siamese fighting fish, also known scientifically Betta sp. is a popular ornamental fish in Thailand. Ornamental fish is a large commercial fish export for Thailand especially Betta sp., which is the top two in term of revenues. There are numerous research paper conducted by Thai researchers to improve the production and conservation of this species. This paper focus on reviewing the general aspect of breeding Betta sp., newer scientific research on genetics of this species, conservation, and which areas should be prioritized by biologists and breeders.

Material and Methods


In Thailand, there are 10 wile Bettas types which are divided into two egg brooding cares of nest building and mouth brooding.

(a) Bubble-nest builder: B. splendens (Regan, 1910), B. smaragdina (Ladiges, 1972), B. imbellis (Ladiges, 1975), and B. sp. Mahachai (has not been classified)

(b) Mouth brooder: B. prima (Kottelat, 1994), B. simplex (Kottelat, 1994), B. pi (Tan, 1998), B. pallida (Schindler & Schmidt, 2004), B. apollon (Schindler & Schmidt, 2006), and B. ferox (Schindler & Schmidt, 2006).

The DNA sequence below illustrates the genetic difference of some of the Betta sp. and Gouramis (Tricopsis and Trichogaster).

The distribution of these wild Bettas as shown in both map below. Fishes are generally found in Paddy fields, marshes, ponds, lagoons, lakes, acidic swamps, streams, and brackish waters. While divulging exact locations may lead to poaching, the are several actual causes that endangered Betta sp. Urbanization, tourism, and agriculture are the main threats to the wild Betta sp. population.

What you're going to do with the knowledge you've gained?
(1) Atison Phumchoosri is a prominent world recognized Thai Betta sp. breeder which have been raising Betta sp. in a grand scale (20000 till 200000 fishes at a time) for many years. As a hobbyist, we don't breed in such scale, but his Thai breeding method is something we can learn from.

(2) Obtain other wild Bettas in MY and try to breed it and compare it with commercially breed Betta sp. and see what the differences. Try look for mouth brooder species.

(3) Investigate whether mangosteen (Garcinia mangostana) have effect on male/female ratio.

(4) Besides tropical almond leaf (Terminalia catappa), Yucca plant can be used in breeding tank to get rid of body water like ammonia. Both these plants extract is the active ingredients in Atison's Betta spa by Ocean Nutrition. Investigate on Yucca plant extract as a complementary ingredient when setting up a breeding environment.

(5) Look into publications and researches done by Horst Linke, the famous aquarist.

What are the further unsolved questions?
(1) Is there a distribution map of wild Bettas species in MY?

Research Paper to Read: (1992) Culture Techniques of Moina: The Ideal Daphnia for Feeding to Freshwater Fish Fry

Source: Science Direct or IFAS.
This is not a research paper in the traditional sense but a published guideline from fishery experts.

What are the key point here?
(1) In term of size, adult Moina is on average is around 700 - 1000μm and for young Moina, average around 400μm. A small size Moina is only slightly larger than Rotifier and smaller than Brine Shrimp. For that reason, Moina is preferable compare to Brine Shrimp as it can live longer in freshwater. (Note: Which is why some Betta sp. breeders only use Moina as the only live feed for all life cycle of the fish)

(2) As Moina is tolerant to poor water quality, it's generally found in any water sources (pools, ponds, lakes, ditches, slow-moving streams, and swaps) polluted with sewage.

(3) Moina strives in temperatures between (24°C - 31°C) but also can tolerance in extreme temperature of (5°C - 31°C) which is suitable for culturing in most part of the world.

(4) Moina feeds on several food sources of bacteria, yeast, phytoplankton, animal manures, detritus (decaying animals or plants), and blue-green algae Microcystis aeruginosa.

(5) Moina reproduces either in sexual or asexual (more common) way. Sexual maturity is around 4 - 7 days with brood size of 4 - 22 per female every 1.5 - 2 days. Each female can produce around 2 - 6 broods in its life span.

(6) Population wise, Moina have higher density (19000 individuals per gallon or 5000 per litre) compare to Daphnia (1900 per gallon or 500 per litre). Production density of Moina depends on food sources. Cultures fertilized with yeast and ammonia nitrate yields 106g/100m3  and compare to Daphnia with similar food source, 25 - 40g/m3. Meanwhile, Moina culture fertilized using organic fertilizer yields even higher output of 375g/100m3.

Why this paper?
The importance of Moina and Daphina as live feed have been discussed in separate post and I will not go details into that. As mentioned, this paper serves as a good introduction and guideline to Moina, the species itself, and most importantly, the culturing techniques in maintaining a Moina population. Moreover, this paper also suggested that Moina is a better live feed compare to Daphnia and Brine Shrimp due to its reproduction volumes and life span in freshwater.

Material and Methods
If you need specific quantity of Moina per day, the suggested method is to batch culture. In batch culture, a new culture is started every day in a separate container. Harvesting should be done around 5 - 10 days where all the food have been consumed by the Moina. Starting new culture every day have another benefit where isolation can prevent contamination due to other Zooplankton or predators.

The previous culturing method combines Moina's food source and its population. Another approach is to use separate culture of food source and Moina population. There are two tanks, the food source or Phytoplankton/algae tank and the Moina culture tank as illustrated below. This setup have the advantages of reducing contamination and sharing of algae tank with multiple Moina tanks. However, extra spaces is needed for such layout.

The next part is on the discussion of the setup and equipment. We start with container, water, aeration, and feeding or fertilizing. Then we proceed with inoculating, monitoring, and harvesting.

38 litres culture tank can only produce enough Moina for hobbyist. For mass production, larger and multiple containers should be used. Water height for the tank should be optimally around 0.4 - 0.5m and can be stretched till maximum of 0.9m. No direct sunlight over the tank and should be some shades to reduce the sunlight till 30% till 50%. To clean up the tank, it should be disinfected with 30% solution of muriatic acid or sun dried.

As Moina is very sensitive to water content, extra care should be taken to prevent poisoning the culture. Anything that contains pesticides, metals, detergents, or bleaches should be removed or discarded before starting the culture. Different water sources have different treatments as shown below:

(1) Well water: aerated for two hours.
(2) Tap water: aerated for two days together with dechlorizer.
(3) Natural spring water, rain water, lake water, or stream water: use as it.

The water temperature in the culture tank should be kept optimally around (24°C–31°C). Anything out of these range will reduce the production of the culture.

Aeration oxygenates the water, suspends food particles, and increases Phytoplankton productions. Apply gentle aeration with no small bubbles as it can cause Moina to trap under the carapace which make it float at the surface and thus killing it.

Feeding or Fertilizing
Different culture medias affects the production of the Moina colony and can be categorized into organic fertilizers and mineral fertilizers. For a 397 litres of water, the quantity (Note: I only keep the metric measurement) that should be added initially for the culture for each fertilizer is shown below. Repeat and replace 50% or 100% of the same ratios after 5 days later.

(1) Yeast: 8.5 - 14.2g of baker's yeast.
(2) Yeast and mineral fertilizer: 8.5 - 14.2g of yeast, and 14.2g of ammonium nitrate.
(3) Alfalfa, bran, and yeast: 42.5g of alfalfa pellets or meal, 42.5g of wheat or rice bran, and 8.5 g of yeast.
(4) Cow manure or sewage sludge, bran, and yeast: 142g of dried manure or sewage sludge, 42.5g of wheat or rice bran, and 8.5g of yeast.
(5) Cow manure or sewage sludge, cotton seed meal, and yeast: Use 142g of dried manure or sewage sludge, 42.5g of cotton seed meal, and 8.5g of yeast.
(6) Horse or cow manure or sewage sludge: Combine 567g of dried manure or sewage sludge.
(7) Chicken or hog manure: Combine 170g of dried manure.
(8) Yeast and spirulina powder: 6g baker's yeast, 3g spirulina powder. Mix with warm water and let it sit for 30 minutes. Pour the mixture through a net into the Moina culture to filter out unresolved yeast.

For hobbyist, the culture medias of item (1), (2), (3), and (8) is more suitable and readily available, especially for indoor environment.

This process is to introduce organisms, in this case, Moina into a culture medium. Typically we call this a Moina starter culture. Start with 100 Moina per litre. Typically inoculation starts after 24 hours or more after fertilization except for yeast with aeration. We can add the Moina after a few hours.

How do we make sure that the Moina culture is healthy and if not, proceed with some adjustments? There are four ways:

(1) Extract one tablespoon or 15ml from the culture and examine with magnified glass or microscope. Pay attention to the colour, intestinal tracts, and movement. In a healthy culture, Moina are green or brown-red in colour with full intestinal tracts and active movement. Unhealthy culture due to environment or insufficient food will produce Moina pale in colour with empty intestinal tracts or resting eggs.

(2) Extract one teaspoon or 3 - 5 ml sample and kill it with 70% alcohol solution. Count the Moina using magnified glass or microscope. If there are 45 - 75 Moina, then the culture is ready for harvesting.

(3) Slightly cloudy, tea colour, or green colour water means there are still food source available for Moina. Only clear water, with transparency of 0.3 - 0.4 m, indicates insufficient food available and need to be replenished with 50% - 100% initial quantity when fertilizing.

(4) Discard the culture and start a fresh new one if the current culture have been infected with predators like Hydra, back-swimmers, diving bettles, or dragonfly larvae.

Use netting net with 50 - 150μm mesh or coffere filter to collect and harvest Moina. For semi-continuous culture, only extract about 20% - 25% from the culture tank. After harvesting, do a partial and small water change and stir the bottom sediments while at it. Moina can kept alive in clean water and fridge for several days with some lost of nutrients due to starvation. For long periods storage, harvested Moina can be kept by freezing in low salinity water (7ppt, 1.0046 density) or freeze-drying. While frozen Moina still retains its nutrient value, it will leach out rapidly upon exposure to water. Enzyme is lost within ten minutes. Amino acids and bound amino acids will lost after one hour.



What you're going to do with the knowledge you've gained?
(1) Buy several Moina starter from different breeders or aquarium shops.
(2) Start two small Moina cultures either in indoor and outdoor environment using plant detritus (decaying water organic matter), alfalfa, bran, yeast, and spirulina powder.

What are the further unsolved questions?
(1) What are the available published literatures on mass scale Moina or Daphnia production?
(2) There are many species of Moina and which species is the most suitable for larviculture?
(3) How do we aerate water without create any bubbles?

Research Paper to Read : (2017) Fruit Fly Maggots as Alternative Feed to Improve Siamese Fighting Fish (Betta splendens) Fecundity, Eggs Hatchability and Fry Survivability

Source: MyJurnal

What are the key point here?
(1) Fruit fly maggots can serve as alternative diet for Betta splendens in addition to bloodworms and formulated pellets which yields result with non-significant differences of average hatchability rate of 65% and survivability rate of 69%.
(2) Commercial pellets is not a suitable diet for broodstock for breeding purpose but it's good enought for daily diet consumption.

Why this paper?
It's rare to find local and recent research paper from local university from MY on Betta splendens. Furthermore, we are curious and researching about alternative diets in addition to existing typical diets like Artemia / Brine Shrimp, Moina, Daphnia, Vinegal eel, Blood worm, Tubifex worm, Mysis shrimp, Grindal worm, Flightless fruit flies, and beef heart. Even though, fruit fly maggots were not a common food source for Betta splendens, it's a common and popular live feed sold in aquarium shops here in MY as a live feed for large ornamental fishes like Arowana.

Material and Methods
This experiment uses 9 pairs (18 fishes) of Betta splendens of 4 till 8 weeks conditioned for a month (the broodstock should be around 8 till 16 weeks old). The fishes were fed with commercial pellets (there is no mention of the nutrients values of these pellets) twice daily during that month.

Experiments were done on three types of feed in 3 replicates:
(1) Commercial pellets x 3 pairs. Control group.
(2) Bloodworms x 3 pairs. Frozen bloodworms purchase from local pet shop.
(3) Fruit fly maggots x 3 pairs. Cultured using over-rippen banana left outdoor, eggs collected and hatched in container. Then the hatched maggots were collected and kept frozen in fridge.

The breeding conditions and procedure as follows:
(1) 1 or 2 days aged water with sea Indian Almond leaves.
(2) Male Betta was released first to build bubble nest.
(3) Female Betta was added but was separated in a separate transparent container.
(4) Female Betta was released once the bubble nest was constructed.
(5) Female was extracted once mating was done.

The number of eggs produced by each diet is shown below. We're quite surprised that commercial pellet yielded a higher result than Blood worms. There is no mention of nutrients content of the commercial pellet so it's quite hard to deduce why commercial pellet is better than live food. Nevertheless, the broodstock with fruit fly maggot diet produced the highest number of eggs.

The nutrients content of both live feed as follows:
(1) Blood worms: 52.8% of protein, 9.7% of fat, 0.38% of calcium and 0.90% of phosphorus.
(2) Fruit fly maggots: 40.3% of protein, 29.4% of fat, 0.59% of calcium and 2.30% of phosphorus.

The high fat and phosphorus may contribute to the high number of eggs spawned using fruit fly maggots as diet. However, there is no details on nutrient of the commercial pellet. Therefore, we can't reproduce this experiment and deduce that commercial pellet may not be a better choice than those two live feed.

What you're going to do with the knowledge you've gained?
(1) Learn how to culture fruit fly maggots to test the suitability of it as an alternative diet for Betta splendens for breeding purpose. Similarly, try with bloodworms as well.
(2) Check if we can breed the broodstock of age of 8 weeks or 2 months. Based on our observation, we don't think Betta fishes of 8 weeks have reach sexual maturity. 12 weeks or 3 months is considered just reaching adulthood.

What are the further unsolved questions?
(1) What are the dietary needs for broodstock to ensure fecundity (the ability to mass produce offspring) or reproductive performance?
(2) What are the feeding schedule in addition to the dietary in question (1)?
(3) As we have mentioned before, quite a few Betta splendens breeders sweared by Daphnia as its primary food source from larvae till adult. Comparison of diet should have includes Daphnia and Miona as well. What will be the expected results if both these two live diets were included?

Research Paper to Read: (2011) Masculinization of the ornamental Siamese fighting fish with oral hormonal administration

Source: ResearchGate.

(1) Oral administration of 17a-methyltestosterone (MT) hormone of 3 and 4 mg/kg doses resulted in 100% masculinization or all males within a spawn.

Why this paper?
Discussion with different breeders shared their experiences buying Betta sp. broodstock from neighbouring country (hint: north) that cannot or won't breed. They have long suspected that the broodstock was somehow treated with certain chemicals that make it harder for the pair to mate and spawn. We've read some papers that probably can explain the root cause of the broodstock that can't breed, masculinization. From early stage of larvae, Through exposure to MT hormone that reverse sex in Betta sp. from early stage of larvae, a breeder can control the sex of the fry by turning the spawn into almost all males Betta sp. This allows the breeder to gain more profits as the male Betta sp. fetches way higher prices compare to female Betta sp. in the ornamental fish market.

Material and methods
The research starts with fry production using 4 pairs of Betta sp. of crowntails, veiltails, and spaidtails strain in four separate 15 litres plastic tank. Once eggs have been fertilized it's extracted and removed to a separate bowls. The spawning was done into two sessions, once in January and another one in May where each experiment group produced around 400 fries. For each experiment group, equal number of 80 fries were placed into 5 separate 2 litres cages within a large 160 litres aquarium tank. The experiment designs in shown in the table below.

Feeding were done 6 times per day (assuming it's 4 hours interval) with different feeding post hatching schedule and diet. DPH is referring to days after post hatching.

(1) Experiment A
- 4 DPH till 7 DPH with artemia nauplii.
- Feeds with MT (Classic AAF-F) until 30 DPH
- Feeds with MY (Lucky Star 2) until 56 DPH.

(2) Experiment B
- 4 DPH till 11 DPH with artemia nauplii enriched with red pepper.
- Feeds with MT (Classic AAF-F) until 30 DPH
- Feeds with MY (Lucky Star 2) until 56 DPH.

The experimental group of A0 and B0 is the control group where no treatment was added. Experiment groups of A2, A3, A4, B3, and B4 shows 100% sex reversal. Mortality rates does not have significant statistical difference.

There are quite a number of confusion in this paper that raise many unanswered questions. Bar chart is not a suitable presentation to summarize the result of the experiment compare to a table with figure. It's very hard to visually identify which experimental group have yield 100% sex change to monosex population.

These treatment were used on ornamental fishes instead of farm fish for food consumption. Hence, it is recommended to use cheaper MT hormones to reduce cost and increase profit.

Further Questions
(1) Why the needs of two separate experiment groups done in two separate period (6 months interval)?
(2) Why both experiment groups have different diet and schedule feeding DPH?
(3) Is MT hormone really have 100% sex reversal impacts on Betta sp.? Can we consider that these fishes is 100% male or in between?
(4) What are the ecological side effects if these fishes were released in the wild?
(5) What are the possible legislative issues on using MT hormone?
(6) Does long exposure to MT hormone affects the mortality rates?

Research Paper to Read: (2012) Effects of photoperiod on reproduction of Siamese fighting fish Betta splendens

Source: Research Gate.

(1) Photoperiod of ratio of 16h light : 8h darkness (spring) and 12h light : 12h darkness (summer) yield optimum reproduction and growth.

Why this paper?
As we have limited space, all our Betta sp. breeding projects were done within an indoor environment. For observation and appreciation purpose, the breeding and growing tanks were equipped with typical T5 LED lights. In the past, we kept the light on for 24 hours because we want to maintain the growth of the aquatic plants. Little we realized, it just dawned to us that animals and plants should follow the natural photoperiods just like in the wild. This begs the question, what is the optimum photoperiods when breeding and keeping Betta sp. Hence, the relevant of reading and understanding this paper.

Material and methods
This research includes 30 matured pairs or 60 fishes of super delta strain. On average, the weight and length of male is 1.92±0.28g and 3.84±0.13cm and female is females 3.72±0.18 cm and 1.72±0.23 g. (Note that the female size is roughly the same as male and typically for breeding, the female size should be smaller). These fishes were hosted within a 2 litres jar.

The design of the experiments for 5 treatments and 6 replicates for each pair of fishes as follows:
(1) 8L:16D
(2) 12L:12D
(3) 16L:8D
(4) 20L:4D
(5) 24L:0D

The breeding conditions for the next 150 days or 5 months are:
(1) Fluorescent light of 60 cm with 200 watt around 500-lux controlled by analogue timer.
(2) Total water changes every two days.
(3) Feeding done twice daily using diet of 45% crude protein and 12% fat in light photoperiod.
(4) Water treatment conditions are: pH 6.9±0.3, dissolved oxygen >5.0 mg L,-1 and ammonia <0.1 mg. L-1. Water parameters are monitored weekly.

The graphs below shows that photoperiod of 16L:8D and 12L:12D shows the significant differences in frequency of spawn, eggs volumes, and fecundity. 24L:0D yields the worst performance of all the measurements.

Does long exposure to light stresses the fishes which affects the production of the spawn size? The result indicates that this may be the main reason. Long light exposure is not natural and does not simulate a natural photoperiod (12L:12D) in outdoor or in the wild. Therefore, extra care needed to control the photoperiod as close as possible following the nature.

Further Questions
(1) Can we obtain similar result if the design and experiment was done in the outdoor environment where the photoperiod was controlled by exposure to sunlight?
(2) What kind of exposure to sunlight is suitable enough? The general consensus among breeder is that you cannot expose the fishes to direct sunlight but within the shaded area.

Research Paper to Read: (2016) Dietary crude protein levels for juvenile beta

Source: ResearchGate.

(1) The range of 30% till 33% crude protein (CP) level is the minimum acceptable level to ensure optimal growth and feeding efficiency for juvenile Betta fish.

Why this paper?
While breeding juvenile Betta fish, instead of live food, there are numerous formulated feeds in the market that we can use. Different brands have different formulation and CP level. What are the acceptable range of CP level to ensure optimal growth and reduce wastage?

Material and Methods
This experiment includes 192 juvenile Betta fish of 30 days age with initial average weight of 0.11 ± 0.02g. These fishes were grouped into 6 groups of CP level with 4 repetitions where each tank contains 8 fishes as shown below:

(1) 27% CP x4 repetitions
(2) 31% CP x4 repetitions
(3) 35% CP x4 repetitions
(4) 39% CP x4 repetitions
(5) 43% CP x4 repetitions
(6) 47% CP x4 repetitions

The breeding conditions for the next 30 days as follows:

(1) 6 litres tank with biological filter.
(2) Airting with temperature 26 ± 1°C.
(3) Feeding (done ad libitum) three times at 07:00, 13:00, and 17:00)
(4) Tank siphoned weekly. (Didn't mention percentages of water changes)

The diet was self-produced formulation feeds using the ingredients shown in table below. The feed was produced by fine grounding the ingredients and mix manually with water around temperature of 50 ± 5°C for starch gelatinisation. Pellets were produced using a meat grinder and dried using oven for 24 hours at 50°C.

As the table below shown, CP of 31% and 35% have the highest weight gain and growth rate. Excess CP slow down growth due to extra energy need to catabolise additional protein and amino acids.

Different fishes have different biological structure and diet needs. Hence, different fishes needs different CP levels. There was one master breeder which overfed the Betta fish to speed up the growth for competition. In his experience, overfed may also lead of loss of fishes as shown in previous table.

Further Questions
(1) What is the relationship between CP percentage and feeding frequency?
(2) What are the CP level needed for adult Betta fish or fry?

Research Paper to Read: (2004) Effect of feeding frequency on growth and fecundity in an ornamental fish, Betta splendens (Regan)

Source: ResearchGate.

(1) Two meals per day is the optimum frequency for growth, gonad (a testis or ovary) development, and fecundity (fertility on producing eggs).
(2) Don't overfed (three times per day) your female Bettas as it will increase it's mortality rate and reduce eggs production.
(3) 14 days is sufficient enough for subsequent spawn for a female Betta.

Why this paper?
One of the question when rearing and breeding Betta splendens is how frequent we should feed the fish to achieve optimum growth and speed up breeding. There are many inconsistent advice given by different breeders on the number of times the fish should be fed. Nevertheless, going through scientific literature will give us a baseline to feed our fishes at the optimum level without unnecessary food waste. However, this does not discounts the vast years of experience of master breeders as typical scientific research was done once compare to years of multiple trials and errors of some breeders.

Material and Methods
This experiment includes 375 juveniles Betta of 30 days old with weight of 0.045±0.01 g and lenght of 14.43±2.2 mm (not sure full length of just body only). The fishes were grouped into 15 groups of 25 each with three sets of each feeding frequency as shown below:

(1) One meal in three days (at 6:00).
(2) One meal in two days (at 6:00).
(3) One meal in one day (at 6:00).
(4) Two meals a day (6:00, 18:00).
(5) Three meals a day (6:00, 12:00, 18:00).

Breeding conditions for 77 days, roughly 2.5 months.

(1) 110-l circular cement cistern with measurement of 53.34 x 45.72 cm (the rounded container typically used by Thai Betta farm).
(2) 50l unchlorinated well fresh water. Tank were drained bi-weekly (didn't mentioned how much water)
(3) Temperature averaged 28±1C,
(4) pH 7.8±0.05.
(5) Water hardness 316±15 mg CaCO3/l, ammonia 1.01±0.12 mg/l and DO 4.04 ppm.
(6) Fresh minced beef liver (protein 35.44%, fat 3.86%, ash, 13.91%, and nitrogen free extract 46.79%)

Calculation of each type of measurements as follows:

(1) Growth. Calculation initial dry weight using electrical monopan balance. Five fishes were sacrificed (not sure how). Weight is determined every 14 days.
(2) Gonad. Calculation initial gonad weight using electrical monopan balance and gonadosomatic index (GSI). Three female fishes sacrificed (not sure how) every 14 days.
(3) Spawning. Upon attaining sexual maturity (did not mention when), two pairs of male and females where selected to spawn in a plastic container with water depth of 15cm. Eggs (hatched or unhatched) counted using sterilized needle.

While the growth rate was expected for those with higher frequency feeding, there was no significant differences between two meals and three meals per day. Interestingly result of spawning size. As the result below shown, if the female Betta was overfeed (three meals per day), the eggs production will decrease. We're quite surprised that second and third spawn yields even more eggs after every 14 days.

It seemed what we did with our breeding project was significantly not helpful to our female Bettas. To increase eggs productions, we've fed both male and female Bettas four meals per day. The result have shown that overfed actually decreased the eggs production and can cause bloating and dropsy. We've observed some of our female Bettas experienced such symptoms after mating.

Further Questions
(1) The paper did not address the impact of different meal plans for juveniles fish from 1st till 30th day. It only accounts for fish starting for 30 days old.
(2) Why not jar each juveniles fish and measure its growth, gonad development, and fecundity individually in separate container instead of one large 110-l circular cement cistern.
(3) To prevent bloating, some breeders skip Sunday meal so that the fish can fast to clear out all its waste. Will this have any significant differences to the result for those 3 meals per day?

Research Paper to Read: (2018) Minimal water volume for intensively producing male Siamese fighting fish (Betta splendens Regan, 1910)

Source: Research Gate.

(1) 150 mL water volume within 2 - 9 cm depth should be the minimum and optimum water volume to rear individual male Siamese fighting fish.
(2) Jar the fish when it reaches 1.5 months old (6 weeks) and sell it at 4 months old (16 weeks).

Why this paper?
Betta fish (B. splendens) is an aggressive and territorial tropical fish. To defend its territories, a Betta fish will flare its gill and spread its fins. Furthermore, it will bite its intruder. Therefore, upon reaching adulthood, the male Betta will need to be separated and jarred into separate and smaller container. However, a Betta fish spawn, on average, contains few hundreds of fries. Hence, to fully utilize available spaces and giving the Betta fish the optimum growing environment, what is the minimum water volume or container size that can achieve this? Our observation is most breeders will use several cost saving ways to jar individual male Betta fish. These consists of using recycle mineral water bottle, thin glass alcohol bottle, or drinking plastic bags.

Material and Methods
This is a randomized design experiment of 5 water treatments and 15 replicates. First, one-month old solid-red male Siamese fighting fishes were purchased and individually acclimatized in cylindrical plastic breakers (7.5cm diameter x 12.5cm height) of water volume of 250mL for 2 weeks (not sure why?). Then, 15 fishes (n = 15) of similar size (0.97 ± 0.01 g initial body weight)were evenly and individually distributed into 5 water volumes of 100, 150, 200, 250, and 300 mL glass aquaria (3.5cm width x 8cm length x 20cm height).

Breeding conditions for 8 weeks or 2 months as follows:
(1) Commercial floating pellets (10% moisture,  46% crude protein, 6% crude lipid, 5% crude,
fiber, and 12% crude ash).
(2) Feeding done twice daily (08:00h and 17:00h) at 2% of body weight.
(3) Photoperiod of 12h (light) : 12h (dark)
(4) Uneaten excess diet were siphoned after 30 minutes after feeding.
(5) 80% water changes with dechlorinated stock water within 3 consecutive days.

Since it's too long and quite complicated (I don't really understand it), I skipped the part on what measurements and tools used.

Only the results for water quality and overall growth were discussed here.

Since leftover food were siphoned, the only contributing factor to water quality is the ammonia level due to excreted water by the fish. The lower the water volume, the higher concentration of ammonia level and lower pH level as shown in table below for water volume of 100mL.

Large water volume (300 mL) will lead to bigger fish growth (standard length) and at the same time, allows more space for increase movement (like bubble nest making) within the container, and thus, low weight gain.

How big is 150mL? The video below will give you some idea. Should you use this minimum water volume for optimum growth? Depends. If you have large quantity of fishes, limited spaces, and plenty of time for water changes, then this should be the right option. If not, use the largest water volume possible so less than frequent water changes. When breeding and rearing Betta fishes, water changes is the only task that consumes most of your time.

Further Questions
(1) Will plastic bottle instead of glass aquaria have any significant effects on the experiment?
(2) When do we start to jar the male Betta fish? Is it 1.5 month of age when it starts to show aggression behaviour or when it reaches certain size of 2.5cm?
(3) Will there any significant changes if the water were treated with Indian Almond leaves?
(4) Is there any significant impact to the result if these fishes were fed with live food sources?
(5) Large water volume means less frequent water change. Is there any impacts on frequency of water change to the result?

Research Paper to Read: (2010) Growth and Survival of Siamese Fighting Fish, Betta Splendens, Larvae at Low Salinity and With Different Diets

Source: Research Gate.

(1) Combination of natural live foods (Chlorella sp., Rotifers, and Artemia nauplii) ensures good growth rate.
(2) Salinity of 5 ppt using non-iodized salt prevents the Piscinoodinium sp. parasite which is the causative agent of velvet rust disease.

Why this paper?
Betta fish (B. splendens) is one of the most popular ornamental fish and it's known to be easily breed. While there are numerous information (websites, books, and videos), most of these information are experiences gained through casual observation rather than scientific research. Two questions were raised during our breeding projects, how can we accelerate the growth and increase the survival rate of our Betta fish larvae (still have yolk-sac and unable to feed themselves) and later as fries (free swimming and can feed themselves)? This paper was written to answer both questions by investigating the best nutrients for the Betta larvae and fries for the first 15 days.

Materials and Methods
The larvaes and fry were the results of a pair of sexually mature (not sure how many months) B. splendens (not sure which species). The pair was fed twice daily (anytime) with flake food and live brine shrimp. Larvae were jararred at 3rd day into 18 round-bottom glass flasks (2L) where each flask contained 10 larvae. These were duplicated into 3 sets as follows:

(1) Treatment: Natural, Salinity: 0 ppt
(2) Treatment: Natural, Salinity: 5 ppt
(3) Treatment: AD + LA, Salinity: 0 ppt
(4) Treatment: AD + LA, Salinity: 5 ppt
(5) Treatment: SDLA + AD, Salinity: 0 ppt
(6) Treatment: SDLA + AD, Salinity: 5 ppt

Culture condition of the larvae as follows:

(1) Salinity was obtained by adding non-iodized cooking salt (aquarium salt should works as well) in portion of 5g/L in distilled water (all minerals were removed).
(2) Temperature set to 26C. (How do they maintain the temperature constantly?)
(3) Photoperiod, 12h light : 12h dark.
(4) 80% water changes on daily basis.

Below is the exact daily rations grouped into three stages of the larvae or fries lifecycle of 15 days. Larvae were fed four-times per day in a 4 hours intervals (0800, 1200, 1600, and 2000h).

Details of the diet with photos (from Wikipedia) as follows:

(1) Freshwater microalgae Chlorella sp.

(2) Rotifers (Brachionus rotundiformis, B. plicatilis "S type").

(3) Artemia nauplii (Brine Shrimp).

(4) Semi-purified microbound formulated diet (particle size 250–450 μm) with ingredients of (37.4% lipids, 46.2% crude protein and 5.6% ash)

As the table below have shown, natural diet yielded the best survival rate and second in growth rate. The total average length of the fries is more precise to the observed and non scientific measurements of the Betta Growth Table.

Why natural live food diet compare to formulated diet gave the optimum survival rate? Betta larvae or fries naturally eat moving living organisms instead of lifeless static food pellets. Furthermore, 100% survival rate was achieved with natural diet and non-iodined salt (5ppt salinity), which prevent the Piscinoodinium sp. parasite which is the causative agent of velvet rust disease as shown in figure below.

This is another photo (source: Wikipedia) showing the actual infection of the parasit.

I'm not sure why the diet of LA + AD with salinity cultured the largest growth length. Unfortunately, this was not discussed in the paper or did I miss or misunderstood it?

Further Questions
(1) When is the sexually mature age for both Betta male or female fish?
(2) How does the frequency of daily feeding (2, 3, or 4 times) influences the larvae and fries growth?
(3) How to calculate the salinity of 5ppt corresponds to the container size?
(4) How does photoperiod influence the growth, survival, or reproduction?
(5) How many percent of daily water changes needed?
(6) Why we need to perform daily water changes?
(7) Does distilled water essential for reproduction, growth, and survival of larvae or fries?
(8) Does the percentage of protein in formulated diet influences the growth and health?
(9) What is the range of salinity level for breeding Betta?