CN115735819A - Hybrid seed production method of crassostrea gigas and crassostrea vitifolia - Google Patents
Hybrid seed production method of crassostrea gigas and crassostrea vitifolia Download PDFInfo
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Abstract
The invention discloses a method for producing hybrid seeds of crassostrea gigas and portuguese oysters, and belongs to the technical field of shellfish hybrid breeding. The hybrid seed production method comprises the following steps: (1) parent selection; (2) parent ripening; (3) artificial fertilization; (4) larva breeding; (5) adhesion modification; and (6) cultivating young shellfish. For crassostrea gigas and portuguese oysters which are relatively far in genetic relationship, the invention establishes a set of breeding method of crassostrea gigas artificial hybrid offspring seeds through links of ripening condition control, spawning mode, incubation condition control, larva and juvenile oyster cultivation and the like, creatively cultivates a new hybrid oyster variety with respective advantages of two parents through a unique one-way fertilization mode, and hybrid offspring gonads develop normally, have fertility, high survival rate, normal growth and high growth speed, and lays theoretical practice foundation for crassostrea gigas artificial breeding and large-scale production.
Description
Technical Field
The invention belongs to the technical field of shellfish hybridization breeding, and particularly relates to a method for producing hybrid seeds of crassostrea gigas and portuguese oysters.
Background
Crassostrea nippona belongs to the phylum mollusca, the class of albopic gills, the family of oyster and the genus of oyster, is naturally distributed in the east Asia sea areas of Japan, korea, china and the like, inhabits at the bottom of rocks with the water depth of 5-15m, and has the characteristics of large individual, slow growth, long culture period of adult shellfish and the like. The spawning period of crassostrea gigas is 8-9 months, and the crassostrea gigas is later than that of crassostrea gigas in north China, so that the crassostrea gigas can keep higher glycogen content in bodies even in summer, and the crassostrea gigas is superior to crassostrea gigas in taste and nutritional value, and is often regarded as a substitute of crassostressostrea gigas in summer. The great market potential endows the crassostrea gigas with extremely high economic value, and in Japan, the market price of the crassostrea gigas in summer is more than 5 times that of crassostrea gigas.
The Ostrea gigas (Crassostrea angulata) belongs to the phylum mollusca, the class of Parabranchia, the family of Ostreaceae and the genus of Crassipes, has the characteristics of small individual, high growth speed, short culture period, thin shell and the like, is widely distributed in coastal areas in south such as Fujian, guangdong and the like, and shows that the yield of Ostrea gigas in China in 2021 is 580 ten thousand tons according to data of the statistical annual book of Chinese fishery in 2022, wherein 38 percent of Ostrea gigas accounts for 210 more than ten thousand tons, and is an important high-yield economic shellfish in China.
Animal or plant crosses between species of the same genus are highly effective breeding strategies. The crossbreeding is widely applied to aquaculture, and is mainly used for improving the growth speed, disease resistance, stress resistance, meat content and survival rate, improving meat quality, creating new varieties, storing and developing beneficial mutants, rescuing extinct fine varieties and the like in the breeding of aquatic animals. In recent years, shellfish hybridization gradually draws attention of breeders and obtains certain results, oyster hybridization can promote interspecific gene exchange, and heterogenous favorable genes are introduced to form a dominant new species.
In actual production, the genetic diversity of oysters is reduced and inbreeding declines due to long-term artificial breeding, so that the development of oyster breeding industry is restricted by the germ plasm resource decline phenomena that the survival rate of artificial breeding is reduced, young oysters often die in a large scale in recent years, and adult oysters are gradually miniaturized. At present, crossbreeding of crassostrea gigas and crassostrea viticola which are relatively far away from each other is not reported.
Disclosure of Invention
Aiming at the problem of germplasm resource degradation in oyster cultivation in recent years and the demand of the market on new oyster varieties, the invention provides a method for producing hybrid seeds of crassostrea gigas and crassostrea viticola.
The invention is realized by the following technical scheme:
a method for producing hybrid seeds of crassostrea gigas and crassostrea viticola comprises the following steps:
(1) Parent selection: selecting large, healthy and harmless and high-activity crassostrea gigas and crassostrea viticola as parents, wherein the crassostrea gigas is used as a female parent and the crassostrea viticola is used as a male parent;
(2) Parent ripening: placing the selected parents in a temporary seawater culture pond with the water temperature of 24-26 ℃ and the salinity of 30-32 for temporary culture for 14-15 days, building a sunshade net above the temporary culture pond for shading, feeding sufficient chlorella twice in the morning and at night every day, and artificially accelerating the maturity of the parents by adopting a micro-aeration method and a micro-flow water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting crassostrea gigas ovum and portuguese oyster sperms respectively, after the collection is finished, placing the crassostrea gigas ovum in seawater with the temperature of 25-26 ℃, the salinity of 25-26 and sand filtration for soaking for 30-35 min, stirring the seawater all the time during the period to prevent the ovum from aggregating and precipitating, then adding the portuguese oyster sperms into the seawater for artificial fertilization, wherein the male-female number ratio of the crassostrea gigas ovum to the portuguese oyster sperms is 1;
(4) Larva breeding: screening D-type larvae, removing abnormal larvae by using a screen, continuing to cultivate, adopting a micro-aeration method in the cultivation process, shading the cultivation, controlling the cultivation temperature to be 25-26 ℃, the salinity to be 24-25, controlling the cultivation density to be within 8-10/mL, feeding baits on the second day after fertilization, feeding golden algae three days before, and mixing chlorella in the golden algae for feeding together three days later;
(5) Adhesion and metamorphosis: observing a proper amount of larvae under a microscope when feeding baits every day, and putting an attaching base when more than 30% of the larvae have eyespots and can see the outwards extending feet;
(6) Juvenile mollusk cultivation: after all the larvae are metamorphosed and attached, continuously culturing in an incubation container for 13-15 days, when the seedling grows to 1-2 mm, moving the seedling into an outdoor cement pond for culturing, controlling the water temperature of seawater in the pond to be 24-26 ℃ and the salinity to be 30-32, mixing and feeding gulfweed, chaetoceros and Platymonas mellifolia, keeping the water color to be light green, continuously feeding and growing the seedlings, and obtaining healthy seedlings.
Further, in the step (3), the number ratio of the crassostrea gigas ovum to the crassostrea portuguensis sperm is 1.
Further, in the step (3), the collection method of crassostrea gigas ovum or crassostrea viticola sperm comprises the following steps: taking out the soft body part of the crassostrea gigas or the crassostrea vitifolia individual, placing the soft body part into a container filled with seawater, controlling the temperature of the seawater in the container to be 25-26 ℃ and the salinity of the seawater to be 25-26, then taking out the eggs or sperms one by one and soaking the eggs or sperms in the seawater, and cleaning the eggs or sperms to remove impurities after all the eggs or sperms are taken out.
Further, in the step (4), before feeding the bait every time, whether the water body is clear or not is observed, if the water body is clear, the feeding amount needs to be increased, and if the water body is not clear, the feeding amount is reduced.
Further, in the step (5), the bait is chlorella, and the bait feeding amount is increased after the attaching base is put in.
Further, in the step (6), the oxygen is increased and the bait feeding amount is increased during the continuous cultivation of the seedling body in the hatching container.
Further, in the step (1), the method for selecting the crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking substances in the gonads of the crassostrea gigas individual, observing the substances under a microscope, selecting the individual with rich substances in eggs and uniform properties as a female parent, and discarding the individual with sperms or hermaphrodite in the gonads.
Further, in the step (1), the method for selecting the portuguese oysters comprises the following steps: selecting a portuguese oyster individual with plump gonad, placing the substance in the gonad under a microscope for observation, selecting an individual with strong sperm motility as a male parent, and abandoning the individual with ovum or hermaphrodite in the gonad.
Further, the micro-aeration method is specifically to utilize an oxygenation pump to aerate air into water to form bubbles, but the water body does not form up-down convection.
Furthermore, the microflow water method is characterized in that a small amount of fresh water is continuously injected through a water inlet, and a small amount of pool water is continuously discharged through an overflow port to form a microflow water environment in the pool.
Compared with the prior art, the invention has the advantages and beneficial effects that:
1. in the invention, the crassostrea gigas is large and grows slowly, the breeding period of adult bivalves is long, the crassostrea gigas is small and grows quickly, the breeding period is short, and the relationship between the two types of bivalves is far. The invention establishes a set of artificial crassostrea gigas fry breeding method through links of ripening condition control, spawning promotion mode, hatching condition control, larva and juvenile shellfish breeding and the like, creatively breeds a new hybrid crassostrea gigas variety with respective advantages of two parents through a unique one-way fertilization mode, is influenced by female parent effect, has the same shapes of shell shape, shell color and the like of hybrid offspring as self-breeding offspring of crassostressostressostressostressostressostresson, but has superior growth speed compared with the self-breeding offspring, and lays theoretical practice foundation for artificial breeding and large-scale production of crassostresson gigas.
2. The filial generation with the advantages of crassostrea gigas and crassostrea viticola can be obtained by the hybridization method, the fertility rate and the hatching rate are higher, the gonad of the hybridized filial generation is normal, the fertility is good, the survival rate of the hybridized filial generation is improved by about 19.57 percent when the filial generation grows to 150 days of age, the shell height is improved by about 35.05 percent, and the total weight is improved by about 117.02 percent compared with the self-breeding filial generation of the crassostrea viticola; compared with the self-breeding filial generation of crassostrea gigas, the height of the filial generation is improved by about 29.82 percent when the filial generation grows to 150 days old, and the total weight is improved by about 64.52 percent; the hybrid progeny obtained by the method has obvious advantages in terms of shell height and full weight compared with the self-bred progeny of crassostrea gigas and crassostrea gigas, provides an effective solution for genetic improvement of crassostrea gigas and the germplasm degradation problem of the crassostrea gigas, particularly the crassostrea gigas in the artificial breeding process in recent years, and has good application prospect.
3. The invention has the key points and difficulties that the fertility rate of gametes of two oysters is improved, the generation of malformed individuals in the process that two oyster hybrid fertilized eggs develop into D-shaped larvae is reduced, and the D-shaped larvae smoothly cross the larval stage. The invention obtains sperms and ova with high developmental maturity by controlling the environmental parameters and the feed types of the parent ripening stage, thereby improving the fertilization rate and the offspring survival rate; the fertilization rate is improved and the deformity rate is reduced by controlling the quantity ratio of sperms and eggs during fertilization, the hatching density and various environmental parameters (seawater temperature and salinity) in the fertilization development process; in the whole larval stage of the D-shaped larvae developing to the eyepoint larvae, the good growth environment and nutrition are improved for the larvae by controlling the breeding environment, breeding density, feed type and feed addition amount of the larvae, so that the growth and survival of the larvae are ensured.
4. The invention strictly controls the environmental parameters and the feed types of the parent in the maturing stage, puts the selected parent in a temporary seawater culture pond with the water temperature of 24-26 ℃ and the salinity of 30-32 for temporary culture, avoids direct sunlight in the temporary culture process, feeds a large amount of chlorella twice in the morning and at night every day, provides good survival conditions for the parent, is favorable for the parent to adapt to the environment of artificial culture faster, promotes the parent to develop stably, promotes the gonad development of the parent more effectively, better spawns and produces sperms in the artificial culture environment, and the sperms grow mature synchronously, provides the ovum and the sperms with better quality for artificial fertilization, and improves the success rate of artificial fertilization.
5. The invention strictly controls the environmental parameters when collecting the crassostrea gigas ovum and the grapevine sperm, when collecting the ovum and the sperm, the seawater temperature is controlled at 25-26 ℃, the salinity is controlled at 25-26 ℃, the soaking environmental parameters and the duration of the crassostrea gigas ovum are strictly controlled, the crassostrea gigas ovum is placed in the seawater with the temperature of 25-26 ℃, the salinity of 25-26 and the sand filtration for soaking for 30-35 min, so that the ovum is activated to reach the optimal fertilization state, the seawater is stirred all the time during the period, the aggregation, the sedimentation and the sperm of the ovum are prevented, and the quantity proportion of the ovum and the sperm is strictly limited, so that the ovum and the sperm are better fertilized, the fertilization rate is favorably improved, and the larva teratogenesis rate is reduced.
6. The invention strictly limits the environment condition of fertilization rate hatching and the hatching density, controls the hatching density of fertilized eggs within 20-25/mL, provides a proper environment condition for smooth hatching of the fertilization rate, and is beneficial to improving the hatching rate. Meanwhile, the invention also strictly controls the cultivation environment, cultivation density and feed variety of the larvae, and the cultivation process is cared for shading, the cultivation density is controlled within 8-10/mL, the bait is fed the second day after fertilization, the golden algae is fed the first three days, the chlorella is mixed with the golden algae and fed together after the golden algae three days later, thereby providing good survival conditions for the growth of the larvae and being beneficial to improving the survival rate of the offspring seeds.
7. The invention strictly controls the environmental parameters and the feed types when the young shellfish is cultivated, increases oxygen and increases the bait feeding amount when the seedling grows to 1-2 mm, controls the seawater temperature of 24-26 ℃ and the salinity of 30-32 in the pond when the young shellfish is cultivated in an outdoor cement pond, and mixedly feeds the gulfweed, the chaetoceros and the platysternon to provide good living conditions for the seedlings, so that the seedlings can grow healthily and continuously, and the higher survival rate of the seedlings is ensured.
Drawings
FIG. 1 is a graph showing D-type larvae produced by the hybridization of crassostrea gigas and crassostrea viticola in example 1.
FIG. 2 is a diagram of hybrid juvenile mollusks of crassostrea gigas and crassostrea viticola in example 1.
FIG. 3 is a comparison of crassostrea gigas and crassostrea viticola with filial generation in the same period.
Detailed Description
The present invention is further illustrated by the following examples, which are provided only for illustrating the present invention and are not intended to limit the scope of the present invention.
Example 1
A method for producing hybrid seeds of crassostrea gigas and crassostrea viticola comprises the following steps:
(1) Parent selection: selecting 20 individual crassostrea gigas and individual crassostrea vitis which are large in size, free of damage and good in vitality from crassostrea vitis population collected from coastal areas of Zhoshan mountain city of Zhejiang province and grape dental oyster population of Zhongpu coastal areas of China as parents, wherein the crassostrea gigas is used as a female parent and the grape dental oyster is used as a male parent;
the selection method of the crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking a proper amount of gonad substances to observe under a microscope, selecting an individual with rich in-ovo substances and uniform properties as a female parent, and discarding the individual with sperms or hermaphrodite in the gonads;
the selection method of the oyster with the portugal teeth comprises the following steps: selecting a portuguese oyster individual with plump gonad, taking a proper amount of gonad substances to place under a microscope for observation, selecting an individual with strong sperm motility as a male parent, and discarding the individual with ovum or hermaphrodite in the gonad;
(2) Parent ripening: in 2021, 9 and 1 days, respectively placing 20 selected crassostrea gigas and crassostrea viticola parents in a seawater temporary culture pond with the water temperature of 25 ℃ and the salinity of 32 for temporary culture for 15 days, shielding the pond with black mesh cloth to avoid direct sunlight, feeding sufficient chlorella twice in the morning and at the evening every day, and artificially accelerating the maturity of the parents by a micro-inflation and micro-flow water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting roe and sperm of Concha Ostreae of Viburnum giganteum at 2021 year, 9 months and 15 days, respectively, soaking roe in sand-filtered seawater at 26 deg.C and 26 salinity for 30min while stirring seawater, preventing ovum aggregation and precipitation, adding ostrea vittata sperms into the sperms, and performing artificial fertilization, wherein the number ratio of ostrea ruttata sperms to ostrea vittata sperms is 1 3 The black hatching barrel is used for hatching, the hatching temperature is controlled at 26 ℃, the salinity is controlled at 25, and the hatching density is controlled within 20/mL, and the D-type larvae are hatched, as shown in figure 1;
the method for collecting the crassostrea gigas ovum comprises the following steps: taking out the soft body part of the crassostrea gigas, placing the soft body part into a container filled with proper seawater, controlling the temperature of the seawater in the container to be 26 ℃ and the salinity of the seawater to be 26, then taking out the eggs one by adopting an anatomical method, soaking the eggs in the seawater, and cleaning the eggs after all the eggs are taken out to remove impurities;
the method for collecting the ostrea grapevine sperms comprises the following steps: taking out the soft part of the Ostrea vittata Linnaeus, placing in a container containing appropriate seawater with temperature of 26 deg.C and salinity of 26, taking out the sperms one by adopting dissection method, soaking in seawater, cleaning after all sperms are taken out, and removing impurities;
(4) Larva breeding: screening D-type larvae, removing malformed larvae by using a 350-mesh screen, continuously culturing, adopting a micro-aeration method in the culturing process, keeping attention to shading, controlling the culturing temperature to be 26 ℃, the salinity to be 25, controlling the culturing density to be within 10/mL, starting to feed baits on the second day after fertilization, feeding golden algae three days before, mixing chlorella in the golden algae after three days, feeding together, observing whether a water body is clear or not before each feeding, increasing the feeding amount if the water body is clear, and reducing the feeding amount if the water body is not clear, according to the situation;
(5) Adhesion and metamorphosis: when feeding bait every day, taking a proper amount of larvae to observe under a microscope, when eye spots of more than 30% of larvae appear and the outwards extending feet can be seen, putting an attaching base, and increasing the feeding amount of the bait after putting the attaching base;
(6) Culturing juvenile mollusks: after all the larvae are metamorphosed and attached, continuously culturing in an incubation container for 14 days, when the larva grows to 2mm, increasing oxygen and bait feeding amount, then transferring the attached substrate into an outdoor cement pond for culturing, controlling the temperature of seawater in the pond to be 26 ℃ and the salinity to be 32, mixedly feeding gulfweed, chaetoceros and flat algae, keeping the water color to be light green, continuously feeding and growing the larvae to obtain healthy larva, and finally obtaining the larva, as shown in figure 2.
Example 2
A hybrid seed production method of crassostrea gigas and crassostrea viticola comprises the following steps:
(1) Parent selection: selecting large, healthy and harmless and high-activity crassostrea gigas and crassostrea viticola individuals as parents, wherein the crassostrea gigas is used as a female parent and the crassostrea viticola is used as a male parent;
the selection method of the crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking a proper amount of gonad substances to observe under a microscope, selecting an individual with rich egg substances and uniform properties as a female parent, and discarding the individual with sperms or hermaphrodite in the gonads;
the selection method of the oyster with the portugal teeth comprises the following steps: selecting a portuguese oyster individual with plump gonads, taking a proper amount of gonad substances to be observed under a microscope, selecting an individual with strong sperm motility as a male parent, and abandoning the individual with ovum or hermaphrodite in the gonads;
(2) Parent ripening: placing the selected crassostrea gigas and crassostrea viticola parents in a seawater temporary culture pond with the water temperature of 26 ℃ and the salinity of 30 for temporary culture for 14 days, shielding the pond by using black mesh cloth to prevent direct irradiation of sunlight, feeding sufficient chlorella twice in the morning and at night every day, and artificially accelerating the maturity of the parents by using a micro-aeration and micro-flow water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting crassostrea gigas ovum and crassostrea davidii sperm, soaking crassostrea davidii ovum in sand-filtered seawater at 25 deg.C and salinity of 25 for 35min while stirring seawater to prevent aggregation and precipitation of ovum, and then adding the ostrea vieniaca sperms into the mixed solution for artificial fertilization, wherein the number ratio of the ostrea vieniaca ova to the ostrea vieniaca sperms is 1 3 The black hatching barrel is used for hatching, the hatching temperature is controlled to be 25 ℃, the salinity is controlled to be 25, the hatching density is controlled to be within 25/mL, and the D-type larvae are hatched;
the method for collecting the crassostrea gigas ovum comprises the following steps: taking out the soft body part of the rock oyster individual, placing the soft body part into a container filled with proper seawater, controlling the temperature of the seawater in the container to be 25 ℃ and the salinity of the seawater to be 25, then taking out the eggs one by adopting an anatomical method and soaking the eggs in the seawater, and cleaning the eggs to remove impurities after all the eggs are taken out;
the method for collecting the ostrea grapevine sperms comprises the following steps: taking out the soft part of the individuals of the grapevine oysters, placing the soft part into a container filled with proper seawater, controlling the temperature of the seawater in the container to be 25 ℃ and the salinity of the seawater to be 25, then taking out the sperms one by adopting an anatomical method and soaking the sperms in the seawater, and cleaning the sperms after all the sperms are taken out to remove impurities;
(4) Larva breeding: screening D-type larvae, removing abnormal larvae by using a 350-mesh screen, continuing to cultivate, adopting a micro-aeration method in the cultivation process, keeping attention to shading, controlling the cultivation temperature to be 25 ℃, the salinity to be 25, controlling the cultivation density to be within 8/mL, starting to feed baits on the second day after fertilization, feeding golden algae three days before, mixing chlorella in golden algae after three days, feeding together, observing whether a water body is clear before each feeding, increasing the feeding amount if the water body is clear, reducing the feeding amount if the water body is not clear, and determining according to the situation;
(5) Adhesion and metamorphosis: observing a proper amount of larvae under a microscope when feeding baits every day, and when more than 30% of larvae are found to have eyespots and can see the outwards extending feet, putting the attachment base, wherein the feeding amount of the baits needs to be increased after the attachment base is put;
(6) Culturing juvenile mollusks: after all the larvae are metamorphosis attached, continuously culturing in an incubation container for 13 days, when the seedling grows to 1mm, increasing oxygen and bait feeding amount, then transferring the attached substrate into an outdoor cement pond for culturing, controlling the temperature of seawater in the pond to be 24 ℃ and the salinity to be 30, and mixedly feeding gulfweed, chaetoceros and platymysternon to keep the water color to be light green, so that the seedlings continuously ingest and grow to obtain healthy seedlings.
Example 3
A method for producing hybrid seeds of crassostrea gigas and crassostrea viticola comprises the following steps:
(1) Parent selection: selecting large, healthy and harmless and high-activity crassostrea gigas and crassostrea viticola individuals as parents, wherein the crassostrea gigas is used as a female parent and the crassostrea viticola is used as a male parent;
the selection method of the crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking a proper amount of gonad substances to observe under a microscope, selecting an individual with rich egg substances and uniform properties as a female parent, and discarding the individual with sperms or hermaphrodite in the gonads;
the selection method of the oyster with the portugal teeth comprises the following steps: selecting a portuguese oyster individual with plump gonads, taking a proper amount of gonad substances to be observed under a microscope, selecting an individual with strong sperm motility as a male parent, and abandoning the individual with ovum or hermaphrodite in the gonads;
(2) Parent ripening: placing the chosen crassostrea gigas and crassostrea viticola parents in a seawater temporary culture pond with the water temperature of 25 ℃ and the salinity of 32 for temporary culture for 14 days, shielding the pond by using black mesh cloth to prevent direct irradiation of sunlight, feeding enough chlorella twice in the morning and at night every day, and artificially accelerating the parents to ripen the parents by using a micro-aeration and micro-flowing water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting crassostrea gigas eggs and crassostrea viticola sperms respectively, soaking the crassostrea gigas eggs in sand-filtered seawater at the temperature of 25 ℃ and the salinity of 26 for 30min, stirring the seawater all the time to prevent the eggs from aggregating and precipitating, then adding the crassostrea viticola sperms into the seawater to perform artificial fertilization, wherein the ratio of the number of the crassostrea gigas eggs to the number of the crassostressostressostressostressostressostressostressostressostrea viticola sperms to the number of the crassostressostressostressostressostressostressostressostressodes is 1 3 Hatching in a black hatching barrel, controlling the hatching temperature to be 25 ℃, the salinity to be 24 and the hatching density to be within 20/mL, and hatching until D-type larvae are hatched;
the method for collecting the crassostrea gigas ovum comprises the following steps: taking out the soft body part of the crassostrea gigas, placing the soft body part into a container filled with proper seawater, controlling the temperature of the seawater in the container to be 25 ℃ and the salinity of the seawater to be 26, then taking out the eggs one by adopting an anatomical method, soaking the eggs in the seawater, and cleaning the eggs after all the eggs are taken out to remove impurities;
the method for collecting the ostrea meretrix sperms comprises the following steps: taking out the soft part of the Ostrea vittata Linnaeus, placing in a container containing appropriate seawater with temperature of 25 deg.C and salinity of 26, taking out the sperms one by adopting dissection method, soaking in seawater, cleaning after all sperms are taken out, and removing impurities;
(4) Larva breeding: screening D-type larvae, removing abnormal larvae by using a 350-mesh screen, continuing to cultivate, adopting a micro-aeration method in the cultivation process, keeping attention to shading, controlling the cultivation temperature to be 25 ℃, the salinity to be 24 and the density to be within 10/mL, feeding baits on the second day after fertilization, feeding golden algae three days before, mixing chlorella in golden algae after three days, feeding, observing whether a water body is clear before each feeding, increasing the feeding amount if the water body is clear, reducing the feeding amount if the water body is not clear, and determining according to the situation;
(5) Adhesion and metamorphosis: observing a proper amount of larvae under a microscope when feeding baits every day, and when more than 30% of larvae are found to have eyespots and can see the outwards extending feet, putting the attachment base, wherein the feeding amount of the baits needs to be increased after the attachment base is put;
(6) Culturing juvenile mollusks: after all the larvae are metamorphosis attached, continuously culturing in an incubation container for 15 days, when the seedling grows to 2mm, increasing oxygen and bait feeding amount, then transferring the attached substrate into an outdoor cement pond for culturing, controlling the temperature of seawater in the pond to be 25 ℃ and the salinity to be 32, and mixedly feeding gulfweed, chaetoceros and platysternon to keep the water color to be light green, so that the seedlings continuously ingest and grow to obtain healthy seedlings.
Example 4
A hybrid seed production method of crassostrea gigas and crassostrea viticola comprises the following steps:
(1) Parent selection: selecting large, healthy and harmless and vitality crassostrea gigas and crassostrea viticola as parents, wherein the crassostrea gigas is used as a female parent and the crassostrea viticola is used as a male parent;
the selection method of the crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking a proper amount of gonad substances to observe under a microscope, selecting an individual with rich egg substances and uniform properties as a female parent, and discarding the individual with sperms or hermaphrodite in the gonads;
the selection method of the portuguese oysters comprises the following steps: selecting a portuguese oyster individual with plump gonads, taking a proper amount of gonad substances to be observed under a microscope, selecting an individual with strong sperm motility as a male parent, and abandoning the individual with ovum or hermaphrodite in the gonads;
(2) Parent ripening: placing the selected crassostrea gigas and crassostrea viticola parents in a seawater temporary culture pond with the water temperature of 26 ℃ and the salinity of 30 for temporary culture for 15 days, shielding the pond by using black mesh cloth to prevent direct irradiation of sunlight, feeding sufficient chlorella twice in the morning and at night every day, and artificially accelerating the maturity of the parents by using a micro-aeration and micro-flow water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting crassostrea gigas ovum and crassostrea davidii sperm, soaking crassostrea davidii ovum in sand-filtered seawater at 26 deg.C and salinity of 26 for 32min while stirring seawater to prevent aggregation and precipitation of ovum, and then adding the ostrea vieniaca sperms into the mixed solution for artificial fertilization, wherein the number ratio of the ostrea vieniaca ova to the ostrea vieniaca sperms is 1 3 Hatching in a black hatching barrel, controlling the hatching temperature at 26 ℃, the salinity at 25 and the hatching density within 23/mL, and hatching until D-type larvae are hatched;
the method for collecting the crassostrea gigas ovum comprises the following steps: taking out the soft body part of the crassostrea gigas, placing the soft body part into a container filled with proper seawater, controlling the temperature of the seawater in the container to be 26 ℃ and the salinity of the seawater to be 24, then taking out the eggs one by adopting an anatomical method, soaking the eggs in the seawater, and cleaning the eggs after all the eggs are taken out to remove impurities;
the method for collecting the ostrea grapevine sperms comprises the following steps: taking out the soft part of the Ostrea vittata Linnaeus, placing in a container containing appropriate seawater with temperature of 26 deg.C and salinity of 26, taking out the sperms one by adopting dissection method, soaking in seawater, cleaning after all sperms are taken out, and removing impurities;
(4) Larva breeding: screening D-type larvae, removing malformed larvae by using a 350-mesh screen, continuously culturing, adopting a micro-aeration method in the culturing process, keeping attention to shading, controlling the culturing temperature at 26 ℃, the salinity at 24, controlling the culturing density within 8/mL, starting to feed baits on the second day after fertilization, feeding golden algae three days before, mixing chlorella in golden algae after three days, feeding together, observing whether a water body is clear or not before each feeding, increasing the feeding amount if the water body is clear, and reducing the feeding amount if the water body is not clear, according to the situation;
(5) Adhesion and metamorphosis: when feeding bait every day, taking a proper amount of larvae to observe under a microscope, when eye spots of more than 30% of larvae appear and the outwards extending feet can be seen, putting an attaching base, and increasing the feeding amount of the bait after putting the attaching base;
(6) Culturing juvenile mollusks: after all the larvae are metamorphosed and attached, continuously culturing in an incubation container for 13 days, increasing oxygen and bait feeding amount when the seedling grows to 1mm, then transferring the attached substrate into an outdoor cement pond for culturing, controlling the temperature of seawater in the pond to be 24 ℃ and the salinity to be 30, and mixedly feeding gulfweed, chaetoceros and Platymonas mellifolia to keep the water color to be light green, so that the seedlings continuously ingest and grow to obtain healthy seedlings.
Comparative example 1
Comparative example 1 is different from example 1 in that comparative example 1 is a method for producing crassostrea gigas and crassostrea viticola by hybridization, which comprises the following steps: the crassostrea gigas ovum and the crassostrea portuguensis sperm are obtained by an anatomical method and are respectively placed in two containers (containing natural seawater which is subjected to sand filtration and is not subjected to salinity adjustment), and the sperms and the ova are directly mixed together for fertilization without controlling the sperm-egg ratio.
Comparative example 2
Comparative example 2 is different from example 1 in that crassostrea gigas produces offspring by self-reproduction in comparative example 2.
Comparative example 3
Comparative example 3 is different from example 1 in that the crassostrea vitis produces progeny by self-reproduction in comparative example 3.
Comparative example 4
Comparative example 4 is different from example 1 in that in comparative example 4, fertilization was performed using ostrea grapevine ovum and ostrea tremula sperm.
Data on the fertility rate and the hatching rate of the offspring were obtained by counting the methods of examples 1 to 4 and comparative examples 1 to 4, and data on the survival rate, the height of the shell and the full weight of the offspring when they grew to 150 days of age were counted, as shown in table 1 below.
TABLE 1 fertilization rate, hatchability, and survival rate, shell height and full weight data for offspring grown to 150 days of age
In the comparative example 1, the traditional method is adopted to carry out hybrid seed production on the crassostrea gigas and the crassostrea viticola, the fertility rate is low, the obtained fertilized eggs have extremely low hatchability, the offspring oysters are difficult to obtain, and the practical production significance is not realized. Comparing example 1 with comparative example 2, the shell height and the total weight of the filial oysters obtained by the method of example 1 when the filial oysters grow to 150 days old are obviously higher than those of the self-bred filial oysters of comparative example 2, compared with comparative example 2, the shell height is increased by 29.82%, and the total weight is increased by 64.52%. Comparing example 1 with comparative example 3, the survival rate, the shell height and the total weight of the filial generation oyster obtained by the method of example 1 when growing to 150 days old are obviously higher than those of the self-propagating filial generation of the ostrea grapevine of comparative example 3, compared with comparative example 3, the survival rate is improved by 19.57%, the shell height is improved by 35.05% and the total weight is improved by 117.02%. In comparative example 4, fertilization was performed using the ostrea viviparous ovum and ostrea tremula sperm, and ostrea viviparous unidirectional fertilization, that is, ostrea viviparous ovum and ostrea viviparous sperm, and ostrea viviparous sperm cannot be fertilized with the ostrea viviparous ovum, so the fertilization rate in comparative example 4 was 0.
FIG. 3 is a comparison of crassostrea gigas and crassostrea viticola with filial generation in the same period, and it can be seen from FIG. 3 that the height and the total weight of filial generation in the same growth period are greater than those of crassostrea viticola and crassostrea gigas.
The comparison shows that the hybrid progeny obtained by the method has obvious advantages in shell height and full weight compared with the self-bred progeny of crassostrea gigas and the self-bred progeny of crassostrea gigas, and has good application prospect in genetic improvement of crassostrea gigas and crassostrea gigas.
Claims (10)
1. A method for producing hybrid seeds of crassostrea gigas and crassostrea viticola is characterized by comprising the following steps:
(1) Parent selection: selecting large, healthy and harmless and high-activity crassostrea gigas and crassostrea viticola as parents, wherein the crassostrea gigas is used as a female parent and the crassostrea viticola is used as a male parent;
(2) Parent ripening: placing the selected parents in a temporary seawater culture pond with the water temperature of 24-26 ℃ and the salinity of 30-32 for temporary culture for 14-15 days, building a sunshade net above the temporary culture pond for shading, feeding sufficient chlorella twice in the morning and at night every day, and artificially accelerating the maturity of the parents by adopting a micro-aeration method and a micro-flow water method to ensure that the gonads of the parents are fully developed;
(3) Artificial fertilization: collecting crassostrea gigas eggs and crassostrea portuguensis sperms respectively, after the collection is finished, placing the crassostrea portuguensis eggs in seawater with the temperature of 25-26 ℃, the salinity of 25-26 and sand filtration for 30-35 min, stirring the seawater all the time during the period to prevent the eggs from aggregating and precipitating, then adding the crassostrea portuguensis sperms into the seawater for artificial fertilization, wherein the number ratio of the crassostrea portuguensis eggs to the crassostressostressostressostressostrea portuguensis sperms is 1;
(4) Larva breeding: screening D-type larvae, removing abnormal larvae by using a screen, continuing to cultivate, adopting a micro-aeration method in the cultivation process, shading the cultivation, controlling the cultivation temperature to be 25-26 ℃, the salinity to be 24-25, controlling the cultivation density to be within 8-10/mL, feeding baits on the second day after fertilization, feeding golden algae three days before, and mixing chlorella in the golden algae for feeding together three days later;
(5) Adhesion and metamorphosis: observing a proper amount of larvae under a microscope when feeding baits every day, and putting an attaching base when more than 30% of the larvae have eyespots and can see the outwards extending feet;
(6) Juvenile mollusk cultivation: after all the larvae are metamorphosis attached, continuously culturing in an incubation container for 13-15 days, when the seedling grows to 1-2 mm, transferring the seedling into an outdoor cement pond for culturing, controlling the temperature of seawater in the pond to be 24-26 ℃ and the salinity to be 30-32, mixing and feeding the gulfweed, the chaetoceros and the platymysternon, keeping the water color to be light green, continuously feeding and growing the seedlings, and obtaining healthy seedlings.
2. The method for producing crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (3), the male-female number ratio of crassostrea gigas ovum to crassostrea viticola sperm is 1.
3. The method for producing crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (3), the method for collecting crassostrea gigas ovum or crassostrea viticola sperm comprises the following steps: taking out the soft body part of the crassostrea gigas or the crassostrea vitifolia individual, placing the soft body part into a container filled with seawater, controlling the temperature of the seawater in the container to be 25-26 ℃ and the salinity of the seawater to be 25-26, then taking out the eggs or sperms one by one and soaking the eggs or sperms in the seawater, and cleaning the eggs or sperms to remove impurities after all the eggs or sperms are taken out.
4. The method for hybrid seed production of crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (4), before bait is fed each time, whether the water body is clear or not is observed, if the water body is clear, the feeding amount needs to be increased, and if the water body is not clear, the feeding amount is reduced.
5. The method for hybrid seed production of crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (5), the bait is chlorella, and the bait feeding amount is increased after the attaching base is put.
6. The method for producing hybrid seeds of crassostrea gigas and ostrea vittata according to claim 1, wherein in step (6), the oxygen is increased and the bait feeding amount is increased during the period of further culturing of the hatching container.
7. The method for producing crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (1), the method for selecting crassostrea gigas comprises the following steps: selecting a crassostrea gigas individual with plump gonads, taking substances in the gonads of the crassostrea gigas individual to observe under a microscope, selecting the individual with rich substances in eggs and uniform properties as a female parent, and abandoning the individual with sperms or hermaphrodite in the gonads.
8. The method for producing hybrid seeds of crassostrea gigas and crassostrea viticola according to claim 1, wherein in the step (1), the method for selecting the crassostrea viticola is as follows: selecting individuals of the ostrea portuguensis with plump gonads, placing substances in the gonads of the individuals under a microscope for observation, selecting individuals with strong sperm motility as male parents, and discarding the individuals with ova or hermaphrodite in the gonads.
9. The method for producing hybrid seeds of crassostrea gigas and crassostrea portuguensis according to claim 1, wherein the micro-aeration method is to utilize an aeration pump to aerate water to form bubbles but not form up-down convection in water.
10. The method for producing crassostrea gigas and crassostrea vinifera hybrid seeds according to claim 1, wherein the micro-flow water method comprises the steps of continuously injecting a small amount of fresh water through a water inlet, and continuously discharging a small amount of pond water through an overflow port to form a micro-flow water environment in a pond.
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