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Pollution-free loach seed breeding methods
**Foreword**
The standard "Pollution-Free Loach" (DB33/561-2005) is divided into three parts:
Part 1: Technical Specifications for Seed Breeding;
Part 2: Aquaculture Technical Specifications;
Part 3: Quality and Safety Requirements.
This section is the first part of DB33/561-2005. It was proposed and managed by the Zhejiang Ocean and Fisheries Bureau. The section was drafted by the Zhejiang Provincial Institute of Freshwater Fisheries and the Xinchang County Fisheries Technology Promotion Station. The main contributors to this section are Zhou Zhiming, Zhang Yuming, Hu Tingjian, and Huang Xiaohong.
**Introduction**
Loach is a highly nutritious fish with a delicious taste, containing up to 21% protein and significant medicinal benefits. As a nourishing food, it has become increasingly popular. However, due to overfishing and habitat degradation, natural loach populations have declined significantly. Artificial breeding has become the primary source of supply, and aquaculture has expanded rapidly both within and outside the province. To ensure a safe and sustainable production environment, promote advanced aquaculture techniques, and guarantee product quality, this standard was developed.
**Pollution-Free Loach: Technical Specifications for Seed Breeding**
**1 Scope**
This section outlines the environmental requirements, broodstock selection, breeding methods, and seed production technologies for loach (*Misgurnus anguillicaudatus*). It applies specifically to the breeding of loach seedlings.
**2 Normative References**
The following documents contain provisions that, through reference in this section, constitute its requirements. For dated references, only the editions cited apply. For undated references, the latest version applies.
- GB 11607: Fishery Water Quality Standard
- GB/T 18407.4: Environmental Requirements for Pollution-Free Aquatic Products
- NY 5051: Water Quality for Harmless Freshwater Aquaculture
**3 Environmental Requirements**
3.1 The production environment must meet the standards outlined in GB/T 18407.4.
3.2 The water source should be clean, abundant, and free from pollution. The water quality must comply with GB 11607.
3.3 Aquaculture ponds should be well-constructed, preferably made of cement or tri-soil. Cement ponds should have a 15–20 cm layer of loam at the bottom. Soil ponds should not use sandy soil. Ponds should be surrounded by cement or plastic walls, 100 cm high, with wire mesh or nylon screens (0.15 mm, 20 mesh) on the inlet and outlet.
**4 Broodstock Requirements**
4.1 Broodstock should be collected from natural spawning areas or from local hatcheries, ensuring genetic diversity and avoiding inbreeding.
4.2 Select broodstock from 2–3-year-old individuals: females weighing 40–100 g, males 20–50 g. They should exhibit typical germplasm traits, be healthy, active, and free from disease or injury.
4.3 Mature females have a swollen abdomen, wide pectoral fins, and a rounded anal fin. Males have a smaller abdomen, narrow pectoral fins, and a protruding area near the genital pore. When pressed, milky sperm should be released.
**5 Broodstock Breeding**
5.1 Stocking density should be 6–10 groups per square meter, with a male-to-female ratio of 1:1 to 1:2.
5.2 Feed twice daily in autumn, using animal-based feeds like minced meat, fish meal, and viscera, supplemented with plant-based feeds such as wheat bran and rice bran. Feed amounts should be 3–5% of body weight. In winter, stop feeding and add fermented pig manure. From late March, increase feeding with high-protein feed at 5–10% of body weight.
5.3 Maintain a water depth of at least 40 cm, regularly replace water to keep the water clear and cool, with a transparency of 30 cm.
**6 Seed Breeding**
6.1 Induction
6.1.1 The breeding season starts from late April to mid-May when water temperatures exceed 22°C.
6.1.2 Use human chorionic gonadotropin (HCG), injecting 500–700 IU per female. Reduce the number of males by half.
6.1.3 Injection sites include the back muscle or dorsal fin. Avoid the abdomen’s midline. Use a No. 4 needle, with 1–1.5 cm exposed. Inject at a 30° angle, 0.2 cm deep.
6.2 Spawning
6.2.1 Optimal water temperature for spawning is 22–28°C, with 25°C being ideal. At 22°C, eggs are laid after 14 hours. At 25–28°C, eggs are laid after 10 hours.
6.2.2 Fertilization
6.2.2.1 After injection, place broodstock in a 150×200×60 cm cage with mesh or brown paper as an inducer. Each group contains 50–80 individuals (male to female ratio 1:2–3). Remove eggs after they are laid and transfer to an incubator.
6.2.2.2 For artificial insemination, gently press the female’s abdomen to collect eggs. Collect male testes and mix with Galinger's solution. Use porcelain bowls and feathers to mix eggs and sperm. After fertilization, rinse with 0.6–0.7% saline and spread evenly on sterilized nests. Incubate in hatching ponds, avoiding direct sunlight.
6.3 Incubation
6.3.1 Fertilized eggs can be incubated in indoor or outdoor microfluidic water. Place egg-filled nests in bamboo baskets and submerge them in water.
6.3.2 Dissolved oxygen in incubation water should be ≥6 mg/L. Other parameters follow NY 5051.
6.3.3 Incubation density is 150–200 × 10ⴠeggs/m³.
6.3.4 Hatching time varies with temperature: 40–44 hours at 22–25°C, 24–28 hours at 25–28°C. After 3 days, when fry change color from black to light yellow, they can be moved to the seedling stage.
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