KR100868894B1 - Method for establishing gonochoristic shellfish selfing line - Google Patents

Abstract

The present invention relates to a breeding method for constructing the offspring lines of male molluscs. The method comprises the steps of selecting a particular individual, ie, hermaphrodite adults, having both female and male germ cells from hermatic molluscs; And constructing and cultivating child lines by a self-correcting mating strategy. Here, the male molluscs can visually recognize the difference in the color of the gonads of the adult adults living by the naked eye. The present invention selects certain variants of male molluscs as breeders for constructing offspring. This facilitates the process of gene homologation and also the process of eliminating harmful genes, thereby promoting selective reproduction in male molluscs. The present invention can be carried out in a simple manner without any particular medicines and devices, and can be easily disseminated. The present invention can be applied to all hermaphrodite molluscs of the living body, in which specific individual features of the hermaphrodite can be visually recognized by the naked eye.

Mollusk, Japanese Scallop, Oyster, Hermaphrodite, Hermaphrodite, Gonad, Color, Self-fertilization

Description

METHOD FOR ESTABLISHING GONOCHORISTIC SHELLFISH SELFING LINE}

FIELD OF THE INVENTION The present invention relates to a breeding method in molluscs, in particular a breeding method for building selfing lines from molluscs of the duodecious.

In mollusks, most species are hermaphrodite, while some are hermaphrodite and some are sexually convertible. Male molluscs generally exhibit a single sexual characteristic at the species level. Sperm, male spouses or eggs, female spouses, can be released from an individual of the male body when they mature. In contrast, the molluscs of the hermaphrodite show positive traits at the species level. When parts of the gonad are mature enough to be recognized, an individual carries both testes, the male gonad, and ovaries, the female gonad. In general, when sexually mature, both sexual partners will be released from individuals of the same hermaphrodite.

In bivalve shells, a giant cockle (a cockle), except for some species, such as giant scallop, bay scallop, etc., has the characteristics of hermaphrodite. ), Blue mussel (Japanese mussels), Japanese scallop, Zhikong scallop, Pacific oyster and manila clam. Almost all species are hermaphrodite. In contrast, in gastropods, despite the higher proportion of hermaphrodite, most of the species exhibiting in vitro fertilization are also hermaphrodite. For example, all species of Haliodae are hermaphrodite.

Hermaphrodite is rare in molluscs of hermaphrodite. Occasionally, both sexual gonads are found, including ovaries and testes, within an individual. A particular feature of these individuals is that female and male germ cells coexist in their gonads when the gonads are mature enough to be recognized. The rate of hermaphrodite varies greatly in different species. In general, this is a slightly higher proportion in oysters, pearl mussels and pearl oysters, but a relatively lower proportion in other species. Haliotis Theuscus Hannah duscus hannai , Pinna pectinata pectinata ), Patinopecen yessoensis ), Chlamys farreri ), Mactra Antiquarata ( Mactra) There are reports of hermaphrodite found in antiquata ) and Phaphia undulta . However, compared with their usual reproductive mechanisms, they were all discussed in abnormal cases. Moreover, all the hermaphrodite individuals were recognized by histological examination when killed. Currently, no reports of hermaphrodite development have been found in the molluscs of the hermaphrodite in their lived cases.

In theory, two techniques can now be applied to the rapid reproduction of homologous lines. Fertility or male reproduction is the most effective method for the gene homologizing process, whereby only one can reach highly homologized genetic material. However, although there are some reports in this area, there are great difficulties in applying them to the breeding of mollusks. At present, female reproduction or male reproduction cannot be applied in the reproduction of mollusks.

Other methods of rapid breeding of allogeneic lineages include child (or self-fertilization), full-sib mating, half-sib mating, Backcross and other inbred-mating strategies are included. Among those mentioned, the above selfing is most effective in the gene homologation process. This is the breeding that occurs among the closest parents among all winter breeding methods. 50% of the specific gene homologation process rate is obtained through one generation and can reach a target gene of homology of 0.984 after six generations. This shows that the process of child gene homologation is faster than some other breeding methods. Therefore, in breeding, it is important to apply the child technology to speeding up the gene homologation process. At present, however, only hermaphrodite organisms can be applied to offspring techniques to speed up reproduction.

As lower organisms, some of the molluscs of the hermaphrodite can cause sex-conversion or hermaphrodite. For these species, it is possible to apply the child, a strategy for building self-producing lines.

As mentioned above, some individuals of the molluscs of the hermaphrodite may undergo sex-conversion during their lives. It is a temporary plan to use cryopreservation of spouses (sperm or oocytes) to generate offspring of these individuals. Single-sex spouses can be obtained by inducing fully mature individuals and can also be cryopreserved. The mature individual is carefully controlled to produce spontaneous opposite spontaneous post-induced spontaneous or artificial (physical or chemical) to cause sex-conversion. Oocytes are spouses of opposite sex, and sperm are mates of these two sex spouses (sperm or oocytes) that are cryopreserved spouses. However, the plan requires two difficult techniques, including cryopreservation and thawing. Moreover, leading artificially induced sex-transfer techniques are not currently realized. Therefore, in order for the plan to be successfully executed, a large amount of challenges need to be solved.

For hermaphrodite molluscs, individuals of that particular hermaphrodite can be intentionally cultured and controlled if they can be recognized in advance during their survival. Both female and male spouses can be obtained simultaneously in a suitable environment. Subsequently, child strains can be achieved by modifying the spouses together. The key to this plan is to recognize living hermaphrodite entities. With the exception of some oysters and pearl oysters, only live mollusks can release these live eggs. In other words, eggs in most mollusk species obtained by dissection do not have enough vitality to develop and do not develop. Only energetic eggs and sperm secreted from these living individuals can be fertilized to build off their offspring. Hence, child lines can be constructed only if the hermaphrodite organisms are known in advance from herma molluscs. However, there were no reports of hermaphrodite individuals being alive in hermaid molluscs.

Pacific avalone ( H. discus) hannai ), P. yessoensis and C. farreri are typical male molluscs. There have been no reports of sex-transformation occurrences or hermaphrodite individuals in these species. According to a survey by the database of BA and Aquatic Sciences and Fisheries Abstracts (ASFA), in 1982 Ryu and his colleagues (Liu et al.) Reported one hermaphrodite of Pacific Avalon; Liao and his colleagues (Liao et al.) Reported one hermaphrodite entity from 241 samples; Similarly, hermaphrodite objects were recognized by Maru Kuniyoshi in the Japanese Scallop. In addition, all hermaphrodite individuals were recognized by anatomical observations killed by dissection during the process of paraffin section, not by examination of the appearance of the gonads by visual or visual examinations.

Typically, male molluscs can possess only one gender gonad, female or male. The gonad is the same color and soft feel at its mature stage. Individuals of different genders in male molluscs can be distinguished from changes in color in the appearance of the gonads by visual or visual inspection. Female or male individuals can be distinguished from the gonad color when they have reached maturity. In molluscs, the ovaries are usually creamy to white, while the testes are relatively more changing colors. Orange (orange), orange-yellow (orange-yellow), go-red (brown-red), Pink (pink color) to the ovary by Calabria Bar River (class Is a non-Tio (Mytioida) and five loop terry in Bivavlia) (Pterioida) are found in mature object in the neck. A Red brown (purple-brown) In; (H. diversicolor small avalone) In Halley five (Haliodae), Pacific Avalon (Pacific avalone) in the ovaries are green gum (dark green), and the small iridescent. However, testes of these species are, without exception, creamy to white.

The inventors of the present invention have successively found some individuals of a particular gonad in Pacific Avalon, Japanese Scallop and Jybean Scallop. Apart from the customary form and soft feel, the gonad is characteristic in its nonuniform color between the ovaries and the testis parts. Both colors of the ovaries and testes were found within the gonads of the same individual, which provides a distinct difference from conventional male molluscs. After complex studies and observations, we examined certain hermaphrodite individuals, ie hermaphrodite organisms, of molluscs, in which two germ cells are present in the gonads. This is the first case of finding and obtaining live hermaphrodite individuals in herma molluscs by observing the gonad portions in the three species mentioned above. However, the study of the offspring of these materials has not been done before. After a series of experiments, we finally developed a breeding method for constructing child lines in male molluscs and completed the present invention.

Summary of the Invention

It is an object of the present invention to provide a breeding method for building the selfing lines in male molluscs. Strategies introduced in accordance with the present invention are as follows.

Strategies to build child lines in male molluscs are the breeders in the gonads as testes (or male gonads) and ovaries (or females) from female molluscs, ie, hermaphrodite. Gonad) Selection of specific individuals with both sex germ cells, followed by application to a method of self fertilization to build and propagate child lines.

The male molluscs mentioned herein visually recognize the differences in the color of the gonads of the individuals or the appearances between males and females from the same species in living state by the naked eye.

The hermaphrodite mollusks referred to herein refer to an individual that is both ovaries and testes in the gonads by examination of the color or appearance of the gonads by the naked eye. Germ cells, including female and male spouses, on the other hand, can be found in the gonads when they are mature enough to be recognized. As indicated by the change in the colors of the ovaries and testes, individuals with a large number of testes and a small number of ovaries are selected as the longitudinal axis for constructing the child lines. In general, the testes should be counted at least 30% or more of the total gonad surface areas; At least the male gonad is visible to the naked eye. The method of screening hermaphrodite individuals as longitudinal axes is carried out in the following procedures: When maturity is reached, each individual is detected by a method of visually recognizing the colors of the gonads. When the human eye recognizes that the sex gonads of both males and females have different characteristics in the color of the gonads, the individuals are defined as specific individuals. These individuals are selected as candidates for hermaphrodite breeders. Subsequently, depending on the degree of gonad maturation of the particular individuals, appropriate conditioning work is performed until the gonads mature enough to build their own lineages. Hermaphrodite individuals can be detected from natural individuals, including wild and raised livestock without artificial induction. Sometimes, if possible, such specific individuals may also be obtained from artificially derived individuals.

Self-correction methods for the construction and reproduction of offspring, as mentioned herein, mean that all hermaphrodite breeders are induced to hatch individually. It is ensured that the female and male spouses from the same hermaphrodite parent have been fertilized. Regular procedures of embryonic, larval and spat rearing are subsequently performed. In the stages of fertilization, embryo, larval and egg rearing, regular separation methods of the offspring are strictly applied to avoid hybridization of other individuals into the offspring.

Detailed procedures are as follows.

Completely mature hermaphrodite by first combining 20-40 minutes, then continuing to immerse the parent (original breeders) in turn in a tank filled with seawater to expose them to thermal shock and UV-irradiated seawater. Adjust artificially to spawn. The sea water was irradiated with ultraviolet light and the temperature was raised to a temperature 3-5 ° C. higher than the conditioning temperature of the longitudinal axes. Observe the scattering of the longitudinal axes carefully. If eggs or sperm are not released within 30 minutes, it is necessary to exchange treated seawater every 30 minutes or similarly until the eggs or sperm are successfully obtained. During the course of the derivation, strict separation of each breeder is strictly required. It is ensured that fertilized eggs and sperm are obtained from the same hermaphrodite.

In some cases, the ratio between ovaries and testes varies clearly in different hermaphrodite parents. The present invention increases or decreases the amount of sperm in order to apply these strategies to produce an appropriate proportion of sperm / eggs. If the testes face a lower rate, separation of testes after normal scattering is performed to promote a high fertilization rate. Conversely, for some hermaphrodite breeders with high proportions of testes, 5-hydroxytryptamine (5-HT) is injected significantly into the organisms by injecting 5-hydroxytryptamine (5-HT) into their organisms. A derivation step can be applied to reduce the sperm that are simultaneously scattered together to induce excess sperm to be spawned. The details are as follows.

Having a lower proportion of male gonads: When male gonads of one longitudinal axis are counted at 0.5% or less relative to the total gonad surface area, the male gonads are considered to be at a lower rate. In this case, when the longitudinal axis scatters, insufficient sperm may be obtained. After normal induction for these individuals, the released spouses are treated as samples observed under a microscope. If more than 10% of the eggs are not enclosed by sperm, they are treated as insufficient sperm. If this case is confirmed, normal derivation is carried out and furthermore even partial dissection of the male gonad of the longitudinal axis is required. The sperm obtained by anatomical means are added in an appropriate amount to the sexual products of the individual which are normally released by normal means. The following details are made, describing this: diluting the sperm with seawater to dilute it to a low concentration (the sperm contained is approximately 10 ⁷ to 10 ⁸ / ml) and diluting the diluted sperm several times Pour into the seawater containing eggs and fertilized eggs of the same breeders, each diluted sperm is 10-50 ml. During this process, the water can be carefully stirred to ensure uniform distribution of sperm. In addition, sampling and testing may be performed every 3 to 5 minutes to ensure that an appropriate amount of sperm is attached to the eggs at one time. In general, sperm are considered sufficient when one or more sperm are attached to 98% of eggs. This behavior is also appropriate when the longitudinal axis of the abundant male gonads distributes insufficient sperm by normal induction.

Having a higher proportion of male gonads: A higher proportion of male gonads means when the male gonads of the longitudinal axis count 15% or more of the total gonad surface area. When these longitudinal axes are guided and spawned in a regular manner, when collected and examined under a microscope, more than a dozen sperm are counted in one eye spot and compared to the amount of eggs that were laid. The amount of sperm released is excessively rich. In order to reach a high fertilization rate, the excessive enrichment of the amount of sperm must be strictly inhibited. The present invention solves this problem by introducing a method of injecting 5-hydroxytryptamine. The amount of 5-HT injected into the muscles of the longitudinal axes is between 100 and 200 μl, after which the longitudinal axes are induced by means of drying in air. The 5-HT reagent is at a concentration of 20 to 500 μmol / L, diluted with sterile seawater, filtered by a micro-hole filter membrane 0.22 μm in diameter. The longitudinal axes injected with 5-HT are impregnated into ultraviolet-irradiated seawater at 3-5 ° C. above the conditioning temperature. Sperm are released about 20 to 40 minutes after impregnation of the longitudinal axes, but in this state the eggs are not released. 200-500 ml sperm of the longitudinal axes are stored individually in a beaker until later utilized for fertilization. 200-500 mL sperm of the breeders can be recovered by dilution with fresh seawater when using them; Residue is discarded. The induced breeders and their containers used are washed with fresh sea water to remove remaining sperm. After 2-4 hours of impregnation of the longitudinal axes, the release of 2-4 consecutive eggs occurs, while a small amount of sperm can also be obtained. After each release of the eggs, the sexual results need to be collected immediately, sampled and examined by microscopy to determine the number of sperm attached to each egg. In batch processing, if sperm are insufficient, it is necessary to add the sperm prepared beforehand by recovery and dilution with fresh sea water. Conversely, if more than 10-12 sperm are attached to every single egg under the indicated observation through the microscope, it is necessary to wash or dilute the eggs with excess seawater. For longitudinal axes, male gonads counted approximately 10 to 15% of the total gonad surface area of the longitudinal axes, the act of injection of 5-HT may be an effective way, which may reduce the number of sperm, High fertilization rates can be obtained and are superior to normal derivation methods.

In addition to the two strategies mentioned above, adjustments in terms of the ratio of sperm and eggs per breeder to the volume and duration of fertilization and fertilization of water are feasible, in a small sense, between the male gonad and the female gonad. Is based on the ratio of the surface areas of. In this act, sampling and microscopic examination are required in time to control the amount of the sperm so that the number of sperm attached to one of the indicated eggs is 10 or less. The principles of this behavior are as follows: When the surface area of the gonads (male gonads), which are cream or colored, is in a low proportion (the surface area of the male gonads counts less than 3% of the surface area of the entire gonads of the longitudinal axis). For example, a small amount (10-20 L) of seawater can be used to induce and correct by extending the fertilization time (20-40 minutes); When the surface area of the gonads, cream to white, is high in proportion (the surface area of the male gonad is counted at least 6% of the surface area of the entire gonad of the longitudinal axis), a large amount (60 to 150 L) of seawater is used. It is also possible to induce and fertilize and also to wash the eggs or dilute the water to induce and fertilize as soon as the spouses are released.

Advantages of the present invention are as follows.

The present invention provides a method for breeding child lines by selecting specific strains having two germ cells among male molluscs as breeders and using the specific strains among male molluscs as breeders. The result is that it promotes the homologation process of genes and also promotes the process of eliminating harmful genes. This promotes selective breeding rates in male molluscs. The invention may be practiced without the need for special medicine and devices. This has the potential to be simple, feasible and easily disseminated. The invention can be applied to the living body of all hermaphrodite molluscs from the characteristics of the particular hermaphrodite objects visually recognized by the naked eye.

Detailed description of the invention

Methods of breeding child lines in male molluscs are described in greater detail below by way of example.

Example  One

The Japanese scallop, Panitofecten Yesohensis, is a typical hermaphrodite. When his gonads are fully matured, it looks like a bull's horn. The ovaries are orange and the testes are cream to white. The sexes can be visually easily distinguished from the appearance of the gonads.

Cultured 12,230 low caliber scallops with mature gonads, obtained from seawater in Liaoning Province, Changhai County, northeast China, were examined in February of the same year. Among the scallops examined, 81 adult bodies were found. The adults with shell heights of 10 to 13 cm had the common characteristics that all individuals had orange and cream to white areas in their gonads, and the cream to white areas for the entire gonad were 0.1 to The change was as high as 80%. The gonad tissues of the two colors are connected, but the boundary of the two colors was very clear. The adults with different proportions of the surface areas of male gonads were selected as longitudinal axes. In the following sections, the breeders were subjected to the growth of larvae as well as spawning, fertilization and cultivation.

1. Holding of breeders

The shells of the hermaphrodite breeders were scrubbed and rinsed to remove superficial (fouling) organisms and deposits. Subsequently, they were soaked in seawater at 5 ° C. (temperature of the surrounding water) and left for 3 hours. The offspring were constructed that day.

2. Selection of breeders

When adult gonads matured, 10 adults with different proportions of male gonads (creamy to white regions) were selected as breeders to stimulate 10 adults to spawn and self-fertilize. Accordingly, ten offspring were constructed, named H ₁ , H ₂ , H ₃ , H ₄ , H ₅ , H ₆ , H ₇ , H ₈ , H ₉ and H ₁₀ . Of these, the parents of the offspring H ₁₀ have the largest proportion of male gonads counted by 50%. That is, half of the parental gonads are cream to white and the other half are orange. The parent of H ₂ , having 15% of cream to white in the gonad, is _second to H ₁₀ . The remaining strains have a lower proportion compared to the two strains and are 3% or less (see Table 1). The creamy to white regions of the parents of H ₁₀ and H ₂ are diffused throughout the gonads. The testes of the other eight longitudinal axes have one common feature. These creamy to white areas are mainly distributed in the gonad near the ventral margins, ie at the top of the horn-shaped gonads or near the side margins.

3. Spawning, fertilization, culture

Shells of the selected longitudinal axes were washed with filtered seawater and then left for 30 minutes in the shade at room temperature. All longitudinal axes were placed in 20 liter plastic buckets each to induce spawning. The distance between each bucket was more than 1 meter. Prior to catching the longitudinal axes, the buckets were filled with 15 liters of UV irradiated, 10 ° C., filtered seawater. After 30 minutes of feeding the longitudinal axes into the seawater, the seawater in the buckets was exchanged with warm seawater once irradiated with ultraviolet light. Care should be taken to observe the release of spouses after placing the breeders in seawater, and in particular to observe the release of sperm. Certain measures were taken following the release of sperm in the water. All ten breeders released most of their spouses within an hour of being introduced into seawater. The spouses released by each breeder contain sperm and eggs (oocytes) at the same time. Immediately after the eggs were released, the eggs were fertilized with the firstborns from the same individuals. For the morphology of the spouses examined by microscope, all germ cells scattered by the ten hermaphrodite individuals were not different from those released by the male gyrus axes. The sperm were strong and active. The yolk particles in the eggs are very large, distinct and evenly distributed. The diameter of the eggs was about 75 μm.

With the exception of the parents of H ₁₀ and H ₂ , the concentration of the sperm of the other eight parents among the sexual products was much lower. The number of sperm around each egg was less than five. Therefore, the practice was relatively simple. Induction of spawning ended after two releases of the eggs in the same way that the longitudinal axes were pulsed. The longitudinal axes could then be taken out of the bucket. During the period in which the released sexual products are held in the inducing water for 40 minutes, the water with ovum and sperm is carefully stirred five times to allow the eggs to be suspended so that the eggs are fertilized close to the sperm. Have the opportunity to do so. Subsequently, the fertilized eggs were diluted directly with fresh seawater to a density of about 40 eggs per ml for incubation at 8 ° C. There is no means to clean the eggs during this period.

For parents of H ₁₀ and H ₂ with relatively large male gonad regions, the following methods were performed during the induction of spawning: due to the nature of the crustaceans that release spouses in the same way as waves, the released products Each wave can be collected and handled separately. After the spouses were released in the form of waves, the products were transferred into a clean 120 liter labeled bucket filled with fresh sea water for dilution. Immediately after formation of the crystal film, the eggs were rinsed to remove residual sperm. At the same time, the breeders and induction vessels were rinsed with fresh sea water and replaced with warm sea water irradiated with ultraviolet light to induce spawning continuously. Careful attention was given to the labeling of the products. The decision of whether or not to keep all batches of fertilized eggs is dependent on the fertilized status of the eggs. Finally, it is time to combine fertilized eggs from the same parent in batches. The batches of products from the parent of H ₂ are five. In batches 1 and 3 of the parental products of H ₂ there are 12-15 sperm around each egg on the same focal plane under the microscope; Within batches 2, 4 and 5 of the product there are 20, 35 and 31 sperm around each egg. The products of batches 1 and 3 were mixed and incubated by diluting to a density of 40 fertilized eggs per ml. Both parents of H ₁₀ released three times. In the third run, sperm dominated in the whole products. After every release, the water was cream to white. The concentration of sperm was very high and they had strong activity, while the eggs were relatively small. The total number of eggs released in three times was 90,000 or less. Hundreds of sperm were found around each egg on the same focal plane under the microscope. The sperm also surrounded the eggs in three layers. All egg caps were hybridized with too many sperm. It was difficult to determine the fertilization rate. Three batches of released products were mixed after the eggs were rinsed.

The mixed product was incubated by diluting to 40 egg densities per mL. However, no D-type larvae were found in the offspring of H ₁₀ . This shows that if the creamy to white gonads (testis) are too large, they can produce too many sperm and therefore the eggs will be difficult to fertilize. However, the sperm of H ₁₀ were used to artificially fertilize the eggs of other male female low-panic Scallop F1, with fertilization and culture rates of 100% and 85.28%, respectively (see Table 1). This demonstrates that the sperm of H ₁₀ have the normal ability to fertilize and incubate. In other words, functionally, spouses of H ₁₀ are normal.

Eggs in each line were between 1,200,000 and 4,000,000. When the fertilized eggs developed to the 2-cell stage, fertilization rates were calculated (see Table 1). Of these, statistical data on the fertilization rate of strain H ₁₀ could not be obtained, and the culture rate thereof was zero. The fertilization rate and culture rate of lineage H ₂ were 68.91% and 24.26%. The fertilization rate of the other eight lines was between 92.4% and 100%, and their incubation rate was 5.86% to 76.25% as calculated at the stage of the D-type larvae.

4. Growth and fixation of larvae and culture of eggs

Larvaes were selected using a 200 μm mesh screen for growth until the embryos developed to the stage of floating D-type larvae. Conditions for rearing in hatchery were 8 larvae / ml and water temperature of 15 ° C. All lines were grown under the same conditions as a whole. Single cell golden algae was used as initial food. After 5 days of growth, diatoms were used as additional food. The horses were fed in such a way that they were quietly added to the water without being scattered four times a day. The seawater in the tanks was completely replaced on a daily basis. Larvae containing tanks were replaced every 5 days. Careful attention has been paid to separation so that the larvae do not mix with different strains during the water exchange and tank exchange processes. If eye drops appeared in 40% of the larvae, seeds were collected using polyethylene nets to collect seeds. After metamorphosis, the amount of feeding increased with the consumption rate of immature scallops. The seawater in the tanks was exchanged half and in total once a day. After 10 days of metamorphosis, the amount and growth rate of the eggs were calculated by sampling the clam cults. Parents of lines H ₁ to H ₉ produced some eggs. The amounts of eggs calculated after 30 days of clam crumbs are shown in Table 1 below.

[Table 1] Ten Child Growth Parameters

system % Of cream to white gonads Amount of eggs (only) % Correction Growth rate (%) Spawning sheep (individuals) H ₁ 0.7 220 94.40 76.25 24000 H ₂ 15 120 68.91 24.26 8600 H ₃ 0.8 260 93.47 73.19 14100 H ₄ 0.9 350 96.67 36.23 86000 H ₅ 0.6 180 92.42 13.33 7900 H ₆ 0.8 260 100.0 29.03 20900 H ₇ 2.6 400 95.83 11.74 16000 H ₈ 0.8 360 95.67 5.86 3800 H ₉ 1.5 210 100.0 21.74 11300 H ₁₀ 50 8.7 Of 0 Of F ₁ * H ₁₀ Of 300 100.0 85.28 Of P Of 500 92.12 40.25 182000  caution ; Lineage P is the control, which was constructed by scattering induction and artificial fertilization of low spaniel scallops of hermaphrodite with normal gonads, and the amount of laying was calculated after providing shell flakes for 30 days.

The aforementioned experiments show that all low-panel scallops with cream to white and orange regions in their gonads are hermaphrodite; It is shown that all hermaphrodite low-scale scallops can be fertilized and develop into embryos, that is, sperm and eggs can produce normal spouses that function normally. This proves that child lines can be built and cultured by the method of the offspring of herpanic low-caliber scallops.

Example  2

The herniated low-panic scallops with large creamy-white areas in their gonads were returned from the external seawater to the hatchery. Their shells were rubbed and rinsed to remove superficial (polluting) organisms and deposits. Subsequently, they were placed in sand-filtered seawater and allowed to stand at a temperature of 6 ° C. for 12 days while supplying the marine stone, Phaeodactylum tricornutum Bohlin . The water was changed half a time twice a day and every three days. H ₂ , H ₂₃ , H ₂₄ , H ₁₀ were hermaphrodite low-caliber scallops with larger cream to white areas in their entire gonads. The ratio of cream to white areas relative to their total gonads was 15%, 20%, 30% and 50%, respectively. H ₂ and the ratio of H ₁₀ is the same as the ratio of the H ₂ and H ₁₀ of the first embodiment. All longitudinal axes were conditioned at 6 ° C. to prepare for induction of scattering. The construction procedure of the offspring was similar to that of Example 1. The main difference was that 5-hydroxytryptamine (5-HT; C ₁₀ H ₁₂ N ₂ O) was used to promote the release of large amounts of sperm before releasing the eggs, thus the amount of sperm in the column of water Effectively reduced. The obvious implementation is as follows.

The longitudinal axes were left to dry in the shade for 30 minutes, and then they were injected with 20 to 500 μmol / L 5-HT. The amount of 5-HT was 100-200 μl (see Table 2). After injection, the scallops were placed in seawater at 10 ° C. irradiated with ultraviolet light to induce spawning. Twenty to forty minutes after soaking in seawater, four pairs of parents began to release sperm. The semen released first in four 500 ml beakers from the buckets containing the four pairs of parents were collected separately and prepared for fertilization. Other sperm were discarded and the parents and induction vessels were rinsed several times. The water was also replaced during rinsing. The release of the sperm of the four pairs of parents was terminated for 40 to 60 minutes each, and the parents and induction vessels were rinsed after every release. After immersion in water for 2 hours, no sperm were released separately by the four pairs of parents. Subsequently, fresh seawater was replaced into the draw tanks every 30 minutes, and all draw containers and parents also rinsed all draw containers and parents when water was changed again. After impregnation into the seawater, within 2.5 to 9.0 hours the four longitudinal axes began to release eggs in the same manner as the wave, but the release of the sperm was greatly reduced. The released eggs were collected in batches. Whether to rinse the eggs or add some more sperm was determined according to the amount of sperm around the eggs. If there are too many sperm around, 10 or more cells are observed under the microscope, the eggs will be rinsed or diluted. If it is observed under the microscope that only about 1 or 2 sperm cells are present around the eggs, the diluted semen already prepared from the same longitudinal axis will be added. To be sure, ① H ₂ 's parents released eggs twice. The first sexual products were added to some semen of the H ₂ parent; The prepared semen of H ₂ was diluted 10-fold with fresh sea water; Subsequently, 10 ml of the diluted semen was taken and added to the fertilized water. Subsequently, the two batches of eggs were mixed and hatched in one tank. ② H ₂₃ 's parents released eggs a total of four times. Excess sperm from the fourth batch of sexual products were discarded. The eggs in the first three batches were mixed to allow fertilization. ③ Due to the excess sperm, all three batches of products released by the parent of H ₂₄ were diluted to 150 ml each, then rinsing the diluted eggs and the first two batches in one tank Incubated by mixing in; The third batch was discarded. ④ Parents of H ₁₀ released eggs for three times. The presence of 15 to 22 sperm around each egg in each batch of eggs was observed under the microscope. Subsequently, three batches of eggs were rinsed each and mixed in one tank to incubate.

All four mixed eggs derived from the four hermaphrodite breeding hatched and were cultured under the same conditions as in Example 1. Experimental results showed that both male and female spouses released by the four pairs of parents were successfully self-corrected except for H ₁₀ ; All three other lines produced immature scallops with an average 600 μm high shell.

The amount of the eggs discharged by the magnetic material of the H ₁₀ was 50,000 or less. The number of sperm around each egg in the three batches was 22, 15 and 20, respectively. The number of sperm of the egg caution was markedly reduced compared to inducing spawning directly without the injection of 5-HT. Due to the lack of eggs, strain H ₁₀ produced little D-type larvae and it was meaningless to continue aquaculture and therefore discarded.

Table 2 Japanese Scallops and their Spouses Injected with 5-TH

system % Of cream to white gonads Concentration of 5-HT (μmol / ℓ) Injected dose (μl) Sperm Release Time (min) Egg release time (hours) Embryos released (batch) The number of sperm in each batch One 2 3 4 H ₂ 15 50 200 40 4.5 2 2 4 H ₂₃ 20 20 200 35 3.0 4 8 6 9 14 H ₂₄ 30 50 100 20 2.5 3 12 14 18 H ₁₀ 50 500 100 30 9.0 3 22 15 20  caution ; The time of all released sperm and released eggs is the first released sperm and eggs; The number of sperm in each batch is the number of sperm around each egg on the same focal plane under the microscope.

Most marine molluscs use in vitro fertilization. In the prior art, for in vitro fertilization, the lack of sperm around one egg affects fertilization rate; Similarly, excess sperm around one egg also affected the culture rate, which significantly increased the number of abnormal embryos and larvae, and even produced hybrid egg layers. Although the ratio of suitable sperm to eggs varies greatly from species to species, it is common perception that the proper number of sperm of attention of one egg on the same focal plane under a microscope is 3-6.

Relatively speaking, the eggs of the Pacific Avalon are less sensitive to the number of sperm around them. 5 to 8 sperm will be very suitable on the same focal plane, but 10 to 15 sperm will not affect hatching. For line H ₂ of Example 1, which was not injected with the 5-HT of the present invention, the number of sperm of attention of each egg in the five batches of sexual products was 12-35 each. However, for the infusion of 5-HT in this embodiment, the number of sperm in each oocyte attention is only 2-4 (see Table 2). This embodiment shows that the injection injection method of 5-HT can effectively facilitate the release of sperm prior to the release of eggs; Thus, it has been shown that it is possible to release markedly reduced sperm simultaneously with eggs. It is also possible to produce offspring by offspring by injection of 5-HT, by the longitudinal axes whose gonads have creamy to white regions of 15-30% in terms of the total surface areas of the gonads. It is shown. However, since little eggs were released by H ₁₀ , there were no offspring from H ₁₀ . Thus, the eggs released by the longitudinal axes with a creamy to white germline area of 30-50% of the total gonad surface area may be in excess of 300,000, and the offspring may be injected by injecting 5-HT to their longitudinal axes. ) There is a good possibility of producing offspring. For adults that are cream to white germline regions equivalent to 10-15% of their total gonad surface regions, the production effect of offspring is best by injection of 5-HT.

Different portions of the germline tissues of H ₂ and H ₁₀ in Examples 1 and ₂ were analyzed by flow cytometry, electron microscopy and paraffin cleavage techniques. All analyzes show that the regions of the gonads are female germ cells while the cream to white regions are male germ cells. Their anatomical structures are the same as those of the conventional Hermaphrodite scallops. Both female and male germ cells at the border of the orange region and the cream to white region were examined simultaneously.

When using the fluid cytometry to examine the creamy to white regions of the gonads of herniform low-pane scallops, there are three peaks of haploids, diploids, and tetraploids, which are mature sperm, secondary hairs. Cells and primary hair cells. This is the same as examining the gonads of male male low-pane scallops in normal males by fluid cytometry. Moreover, when using the same technique to examine the orange regions of the gonads of the herniform low-pane scallops, there are two peaks of diploids and tetraploids, which represent oocytes and oocytes. This is also the same as the flow cytometry of normal females to examine the gonads of hermaphroditism low scallops. When scanning electron microscopy (SEM) is used to examine the creamy-white areas of the low-panel scallops of the hermaphrodite, the structure of sperm is different from that of sperm from normal male hermaphrodite scallops. Did. When using paraffin cleavage techniques to examine the orange region, cream to white region and their boundaries of the hermaphrodite low scallops, the orange portion of the gonads is the normal female hermaphrodite scallops. Identical to tissue, and the creamy to white portion of the gonad was identified to be the same as the tissue of normal male hermaphrodite scallops; Female and male germ cells were examined within the boundaries of the orange and cream to white regions. The aforementioned experimental results indicate that the lower california scallops with two colored portions of the tops of the female (orange) and male (creamy to white) gonads of the lower scallop are hermaphrodite individuals and the orange portion of their gonads This ovary, and the creamy-white part of their gonads is seminal vesicles; It has been demonstrated that their female and male germ cells coexist and develop simultaneously. Thus, the low-profile scallops of the hermaphrodite can be recognized by the color of their gonads.

Example  3

The Japanese scallops H ₃₀ and H ₃₁ had very small male gonad regions. H ₃₀ had only one creamy-white spot on the horn-shaped tip of the bull's horn of his gonads. His male gonad was counted as 0.1% of the total surface area of his gonad. H ₃₁ had a nearly round creamy-white spot on the side of its gonads. The point was counted as 0.5% of the total surface area of its gonads. The conventional method for the induction of scattering was almost the same as in Example 1; After drying for 30 minutes in the shade, scattering was induced in 10 L of preheated, UV irradiated seawater. H ₃₁ was within 35 minutes, while H ₃₀ began to release spouses within 30 minutes. The adults of H ₃₀ and H ₃₁ were withdrawn out of the water after 1.5 hours, and then the water was carefully stirred to allow the eggs to float and to have ample opportunities for binding with sperm. After 10 minutes, sampling and microscopic examination confirmed that H ₃₁ was 85%, whereas H ₃₀ was only 50% of the eggs surrounded by sperm.

The creamy-white spots of the gonads of H ₃₀ and H ₃₁ , respectively, and were incised with a surgical knife, and the wounds were carefully washed with sea water and allowed to pass sperm into 200 ml beakers. When the aforementioned steps were performed repeatedly, the cream-white sperm could also be seen by the naked eye. It poured the total sperm H ₃₀ into the first four times, and two times for 3 minutes while keeping the gap between the two times of the latter released into the conventional sexual products from the ₃₀ H for. During this process, the modified water was stirred to allow the sperm to be uniformly dispersed. Sperm diluted to one-fifth of its original density was poured into its usually released sexual products three times, 20 ml each time, for five minutes at a time.

Based on the available techniques, eggs in the phylum Mollusca can form a fertilized membrane when fertilized, and they become insensitive to sperm. Thus, the experiments of this embodiment of the present invention can be applied to the measurement of increasing sperm in a batch to avoid problems such as abnormal embryos caused by too many sperm.

Example  4

The gonads of C. farreri are very similar to the gonads of the Japanese scallops. After their gonads mature, they look similar to the bull's horns, the female gonad is orange, and the male gonad is cream to white. It is very easy to distinguish gender from the appearance of their gonads. On the other hand, the hermaphrodite phenomena are rarely seen.

In May of a particular year certain individuals were found, mainly orange, from some conditioned scallop scallops. Very small creamy to white spots are distributed in the gonads of this adult. The diameter of the dots was about 0.1 mm. The surface area of all the cream to white spots was counted as 0.2% of the total surface area of its gonads. The subjects were dried in the shade for 20 minutes and subsequently spawned in 23 ° C., UV irradiated seawater. The UV irradiated seawater was exchanged twice after 30 and 50 minutes, respectively, when the adults were impregnated with water. After 10 minutes of exchange of the latter, spouses began to be released. The sexual products released by this adult were averaged and divided into A and B groups. The sexual products of group A were self-corrected without external sperm. Sperm were examined only around 60% of the eggs under the microscope, showing a significant decrease in the number of sperm. However, the sexual products in group B added some sperm scattered by normally induced male gyro scallop so that all eggs were surrounded on average by four sperm. In the four-cell stage, group A had a fertilization rate of 82.1%, while group B had 98.6%. The results demonstrate that the gonad is an individual of the hermaphrodite, which was previously mentioned with a gyro scallop including orange and creamy to white areas. The eggs and sperm produced by the hermaphrodite individuals of Xybean Scallop can be self-fertilized and their spouses can function normally.

Example  5

Pacific Avalon ( H. discus The gonad of the hannai looks like a bull's horn when fully matured. Typically, male and female individuals can be easily distinguished by the color of the gonads. Female gonads show dark green, while male gonads show cream to white color.

It has been found that one of 2300 Pacific Avalon adults that have been conditioned to mature has a gonad of mature, regular but uneven color. It is labeled 231 and its gonad is green except for a small, creamy-white small spot on its central posterior region. The dots appeared square and counted as 5% of the total surface of the gonads. This adult was collected from seawater in Miyagi Prefecture, Japan. The adult was not conditioned to induce scattering until the effective accumulative temperature (EAT) reached 1400 ° C. His shell length was 10.8 cm and his gonads were fully matured. The temperature of the conditioned water was 20 ° C.

The adult was induced to scatter under the following conventional experiments: Drying No. 231 in the shade for 40 minutes; It is separately put into an Avalon-scattering tank; Pouring seawater irradiated with ultraviolet rays at 600 Pa · h / l at 23 ° C. into the tank; After 40 minutes, seawater at 23 ° C. was exchanged once. 50 minutes after being introduced into the seawater, the adults began to release eggs and sperm. This spawning lasted 1.5 hours, releasing 30,000 eggs in total. The adult was removed and the seawater was gently stirred every 10 minutes for the next 1 hour to keep the eggs floating. Subsequently, hatching and breeding were carried out according to conventional steps.

Over 400 crawling larvae survived. Under the microscope, 42 normal and active larvae were harvested for settling. The transplantation was performed in a 10 L avalon-hatcher tank. On the wall of the tank are attached some low-life stone horses. The selected 42 larvae were transferred into the bath. The wall of the tank acted as clam crumbs. The bath was left at room temperature of 20 ° C. and the larvae were grown in quiet seawater exchanged once a day. Three months later, three immature avalons survived. Subsequently, the immature avalons were removed from the wall of the bath and transferred into black corrugated plastic plates and incubated in a conventional manner in a net-cage. These three individuals were grown by April of next year, and their shell lengths reached 1.6 cm, 1.7 cm and 2.1 cm, respectively. The experiment showed that the Pacific Avalon having a gonad with both dark green and cream to white were individuals of the hermaphrodite; They can be induced to simultaneously spawn female and male spouses, further demonstrating that individuals of the hermaphrodite of Pacific Avalons can self-fertilize and obtain their own offspring. have.

According to the present invention there is provided a method for farming the offspring of a male mollusc, which can be recognized by the naked eye as a female or a male based on differences in the appearance of their gonads, and the children of the male molluscs. The method for farming lines can also be applied to molluscs whose eggs can be obtained through dissection. Let's explain using oyster as an example.

Oysters are males with high proportions of hermaphrodite adults, but both female and male gonads are cream to white. Thus, by observing the color of the gonads with the naked eye, it is impossible to distinguish hermaphrodite objects. Interestingly, male and female spouses of oysters can be obtained by dissection. Moreover, their zygotes can be obtained normally by examining the tissues of the gonads of all oysters; Once an individual is found containing both eggs and sperm at the same time, the individual can be separated separately to obtain their male and female spouses and to modify them; It is possible to grow offspring. This solution can be applied to any molluscs that can obtain eggs by dissection, such as oysters, pearl shells, and the like.

Therefore, according to the present invention, there is an effect of providing a breeding method for building the child lines in the male molluscs.

Claims (8)

1) selecting a hermaphrodite adult, a particular individual having both female and male germ cells, from any one mollusc mollusk selected from avalon and a male scallop; And 2) establishing and farming the self-correcting breeding lines of the selected adults by a self-correcting breeding strategy; In step 1), certain individuals of the male molluscs can visually be recognized visually by the naked eye, having both different female and male gonads when their gonads mature. Predominantly, in terms of the surface area of the gonads, the surface area of the male gonads is counted as 30% or less of the surface area of the entire gonads so that the male gonads are observed at least visually; Breeding the self-fertilization mating strategy and offspring in step 2) induces spawning of individual adults, self-fertilizes female and male spouses from the same breeder, and in a conventional manner Incubating, cultivating and growing self-fertilized eggs; Wherein the induction of spawning and self-fertilization is induced in different tanks by means of artificial stimulation, so that each adult releases spouses, and subsequently fertilizes eggs with sperm from the same adult To; Increasing or decreasing the number of sperm around each egg in the same seawater to artificially interfere or control the proportion of both spouses according to the proportion of the two gonads; In view of the surface areas of the gonads, the adults whose male gonads are counted from 15 to 30% of the total gonads are dried in the shade, injected with 5-hydroxytryptamine, and 3 to 3 times larger than the original conditioned seawater in UV irradiated seawater. A breeding method for constructing child lines from male molluscs, characterized by inducing spawning at a high temperature of 5 ° C. delete delete delete 2. The method of claim 1, wherein the act of artificially interfering and controlling the proportion of the female and male spouses induces spawning for the adults in which the male gonad is counted to less than 0.5% of the total gonad in terms of the surface area of the gonad. And then providing sperm released by inducing sperm by partially dissecting the male gonads of the adults. 5. The method of claim 1, wherein the act of artificially interfering and controlling the proportion of the female and male spouses is performed on the adults in which the male gonad is counted from 10 to 15% of the total gonad in terms of the surface area of the gonad. A method of breeding for constructing offspring from said male molluscs, characterized by injecting hydroxytryptamine. 7. The offspring from the male and female molluscs of claim 1 or 6, characterized in that the 5-hydroxytryptamine is injected in the concentration of 20 to 500 [mu] mol / l for each adult. Breeding way to build a farm. 2. The breeding of claim 1, wherein the avalon is Pacific Avalon and the scallop is a low scallop or gyro scallop. Way.