Linpe Method of Induced Breeding

Working together, Canadian and Chinese researchers have now developed a fish breeding method that increases the efficiency of aquaculture production. Injecting a GnRHa followed by (or in combination with) a dopamine antagonist has been called the Linpe method, after Lin, Hao-Ren; Peter, R.E. 1988, the researchers who started it. Most of the work resulting in the Linpe method was done on cyprinids, and there is convincing evidence for these fish that the method is effective where injection of GnRH alone is not. Linpe method — induces ovulation in female fish by injecting them with a combination of a synthetic gonadotropin-releasing hormone analogue (LHRN-A) and the drug domperidone. The hormone stimulates the sex organs of the fish, while the drug inhibits the action of dopamine, a substance produced by the fish that inhibits ovulation.

With traditional fish spawning methods, carp, for example, are raised and killed to produce a pituitary extract used to induce spawning. Many fish are sacrificed in the process and the extract has a poor shelf life. The technique also requires that fish are injected at two separate intervals to induce ovulation.

The new method reduces the cost of production, increases the supply of seed fish, and is more convenient. Rates of spawning, fertilization, hatching, and survival were significantly higher in research trials than could be achieved with pituitary injections. The hormone and drug can be introduced together, which means that brood fish stocks are handled only once, reducing the risk of disease or damage to the fish. This method does not alter the reproductive cycle of the fish, and the fertility and viability of offspring are normal. The solution does not require refrigeration and has a long shelf life. It has been tested on a wide range of fresh, salt, and brackish water species, including carp, bream, salmon, catfish, loach, and others.

It is tempting to generalize about the superiority of the Linpe method for all cultured fish, but because comparative experiments under field conditions - GnRHa-domperidone versus GnRHa alone - have only been done for carps; it is still too early to do this. Some researchers have tried to put fish in categories that reflect the strength of dopamine inhibition, ranging from cyprinids (strong dopamine effect) to salmonids (weak dopamine effect). The danger of making this kind of list relates to what we have already said about differences in the effect of GnRH itself: factors such as the general readiness of the fish can outweigh any advantage or disadvantage of a particular treatment.

The commonly voiced view that marine fish do not require domperidone along with GnRHa requires more proof; in milkfish and mullet, for example, two of the most important warm-water marine species, there are no published reports of its having even been tried. Until use of the Linpe method is more widespread, we will avoid such lists. In species that become fully sexually mature in captivity and respond to GnRHa readily - many salmonids fall into this category - a dopamine antagonist is not needed. In other species - the best evidence is still from cyprinids - even though they will spawn with GnRHa alone, delay to ovulation is shorter and more predictable when domperidone is added. Administering the two drugs is easy, with a single injection of a mixture being as effective as two separate injections. This has led to the manufacture of a commercial GnRHa-domperidone spawning~kit" that combines the two in a single solution (Ovaprim-C@). Enterprising fish farmers can of course always opt, as in Thailand, to buy GnRHa and domperidone as over-thecounter pharmaceuticals, and reconstitute them for injection into fish.

According to Dr. Lin Hao-Ran of China's Zhongshan University, the Linpe method has become "...more and more popular in Chinese fish farms and has replaced the traditional fish spawning methods in recent years". Dr. Lin has established a commercial operation to sell the active compound in China through the Ningbo Hormonal Products Factory in Ningbo City.

Commercialization was identified as a specific objective in Phase II of the research project. In addition, Syndel International Inc. has submitted an application (pending) to register Ovaprim at the regulatory agency in China, after running clinical trials in Wuxi, Beijing, and Harbin in 1994.

Linpe method (domperidone/sGnRH-A) of induced spawning of cultured freshwater fish are used in many countries, for leading to commercialization of the method; to determine the effectiveness of sGnRH-A and domperidone in induced ovulation and spawning of marine teleosts; to determine the effects of aging on reproductive function of key species in the Chinese freshwater polyculture system; to determine means of increasing growth rates of cultured fish and; to continue the training of young Chinese scientists in relevant disciplines.

Doses of GnRH and domperidone

Although there will never be a standard method for spawning all species, culturists working with a single species can standardize methods by systematically eliminating sources of variability and using the lowest effective dose. Effective doses of GnRHa and domperidone vary widely and are not comparable because of differences in species, temperature, state of maturity, and GnRHa. The trend is toward single injections and, although GnRHa doses between 1 and 100 ~g/kg have been effective, culturists should aim for the 5-20 ,~g/kg range. Domperidone is usually effective at doses of 1-5 mg/kg.

To facilitate economical use of GnRHa, without the need for tedious weighing of tiny amounts, it is best to buy preweighed small amounts of the hormone (e.g., 0.5 or 1 mg aliquots) and prepare a concentrated stock solution (e.g., 1 mg/mL) in sterile water in the original container. Appropriate amounts of a more dilute solution in 0.7% NaC1 can then be prepared at the time of injection. GnRHa is most stable as a dry powder, but the sterile stock solution can also be kept for several months if frozen.

Domperidone and pimozide are not readily soluble in water and are sensitive to oxidation. They are best used as a suspension in 0.7% NaC1 containing 0.1% metabisulphate as antioxidant, or can be dissolved (and injected) in propylene glycol. Commercially available domperidone tablets for humans (Motilium0) have been powdered, dissolved in propylene glycol, and used uccessfully in induced reproduction of fish (Fermin 1991).