Method for artificially inducing triploid hybrid of megaloctenus acer and procyon lotor and application thereof

Abstract

The application belongs to the technical field of fish genetic breeding, and specifically discloses an artificial induction method of Megalobranchius ambius and Culter alburnus hybrid triploid and application. The artificial induction method comprises the following steps: S1. taking Megalobranchius ambius as a female parent and Culter alburnus as a male parent to perform artificial dry insemination; S2. performing hydrostatic pressure treatment on the fertilized eggs, and the hydrostatic pressure treatment conditions are as follows: the starting time of treatment after fertilization is 2-3 min, the pressure is 35-50 MPa, and the duration is 3 min; S3. hatching the fertilized eggs and performing transfer culture, and the water temperature in the whole process from fertilization, hydrostatic pressure treatment, hatching to fry cultivation is controlled to be 22-24 DEG C; and Megalobranchius ambius and Culter alburnus triploid is screened through ploidy analysis. The method can successfully induce Megalobranchius ambius and Culter alburnus hybrid sterile triploid with superior growth performance, and based on comprehensive index evaluation, the best hydrostatic pressure treatment condition is obtained, high triploid rate (>80%) and high survival rate (>98%) are simultaneously achieved, and commercialized breeding production can be realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fish genetic breeding technology, and particularly relates to an artificial induction method of triploid hybrids of Megalobrama amblycephala (♀) and Culter oxycephalus (♂) and application. BACKGROUND

[0002] Megalobrama amblycephala and Culter oxycephalus belong to Megalobrama and Culter of Culterinae, respectively. Megalobrama amblycephala is a kind of important freshwater aquaculture species in China, which is omnivorous and has a lower breeding cost and a faster growth rate. Culter oxycephalus is a kind of meat-eating fish with a long and slender body, tender and delicious meat, thin and small scales, and a high breeding cost. Although the two species are quite different in terms of food habits, growth and stress resistance, they have a great trait complementarity.

[0003] Distant hybridization is a method of hybridization between different species or even more distant individuals, which can effectively combine the excellent traits of the parents and increase the variability of the offspring, so as to create excellent hybrid new varieties. At present, the utilization of hybrid vigor of Megalobrama and Culter has been successful in breeding and production: in recent years, China has successively approved six new hybrid varieties of Megalobrama and Culter, which have the advantages of fast growth, good meat quality and strong stress resistance. However, the above-mentioned hybrid new varieties of Megalobrama and Culter can only be bred in restricted water bodies, because these hybrid varieties are fertile, and if they escape during breeding, they will cause great damage to the original species germplasm resources in nature. Therefore, it is necessary to develop sterile triploid of the hybrid new varieties of Megalobrama and Culter.

[0004] The naturally occurring triploid fish such as Carassius auratus can reproduce through gynogenesis and has the ability to reproduce. The artificially induced triploid fish has a odd number of chromosome sets, cannot normally undergo meiosis, and loses the ability to reproduce due to the atrophy of gonads, so it has ecological safety and does not need to be bred in restricted water bodies. Even if it escapes during breeding, it will not cause damage to the original species germplasm resources in nature. There are many methods for artificially inducing fish triploidy, mainly including temperature shock (cold or hot), hydrostatic pressure, chemical drug treatment of fertilized eggs such as colchicine and cell fusion, and nuclear transplantation.

[0005] The above-mentioned artificial induction methods have their own disadvantages: the nuclear transplantation technology is complex and not suitable for large-scale batch production. The chemical drug residues may have an impact on the environment, and the quality and safety of aquatic products cannot be guaranteed. The two commonly used physical methods of temperature shock (cold or hot) and hydrostatic pressure are safer for triploid induction, but the temperature shock method causes great damage to the fertilized eggs, resulting in a low survival rate and difficulty in production and application, while the hydrostatic pressure method has a large difference in triploid proportion and survival rate in different fish. At present, there is no report on the sterile triploid of Megalobrama and Culter hybrids, so how to develop a method for successfully inducing sterile triploid of Megalobrama and Culter hybrids that can overcome technical drawbacks is a problem to be solved. SUMMARY

[0006] To solve the above problems, one of the purposes of the present application is to provide an artificial induction method of triploid hybrid between Megalobrama amblycephala and Culter alburnus, which successfully induces sterile triploid hybrid between Megalobrama amblycephala and Culter alburnus with high triploidy ratio and high survival rate, and can realize commercial breeding production.

[0007] To achieve the above purposes, the specific technical solutions adopted by the present application are as follows:

[0008] An artificial induction method of triploid hybrid between Megalobrama amblycephala and Culter alburnus, comprising the following steps:

[0009] S1. Using Megalobrama amblycephala as female parent and Culter alburnus as male parent, respectively injecting the parent fish with spawning stimulating drugs, and then temporarily raising the parent fish in an egg laying pool while stimulating the development of gonads by flowing water; when the parent fish can squeeze out eggs and sperm, the parent fish is taken out and squeezed to obtain eggs and sperm in a dark environment, and then artificial dry insemination is performed (dry insemination is that the sperm and eggs are mixed without water at first, and insemination does not occur, and insemination begins after water is added, so as to accurately control the start time of insemination) ;

[0010] S2. The fertilized eggs are treated by hydrostatic pressure, and the hydrostatic pressure treatment conditions are as follows: the start time of treatment after fertilization is 2-3 min, the pressure is 35-50 MPa, and the duration is 3 min;

[0011] S3. After the hydrostatic pressure treatment is completed, the fertilized eggs are hatched in a hatching barrel until the fry can swim normally, and then the fry is transferred to a large soil pond for breeding; the water temperature in the whole process from insemination, hydrostatic pressure treatment, hatching to fry breeding is controlled at 22-24℃ (if the water temperature is too low or too high, it will affect the insemination and hatching of the parent fish, thereby reducing the hatching rate; it will also affect the development of embryos and fry, leading to a decrease in survival rate and developmental deformity; it may also cause a decrease in fish immunity, making the fish susceptible to diseases; in addition, the fertilized eggs and fry of fish are very fragile and need a stable environment, so the water temperature needs to be controlled in a relatively narrow range); the ploidy of the hybrid fish is determined by measuring the relative DNA content of blood cells by a ploidy analyzer, and the triploid hybrid between Megalobrama amblycephala and Culter alburnus is screened; the induction effect is evaluated by a comprehensive index, which is calculated by taking the relative hatching rate of 60% and the triploid rate of 40% as weights.

[0012] Preferably, in step S2, the hydrostatic pressure treatment conditions are as follows: the start time of treatment after fertilization is 2.5 min, the pressure is 45 MPa, the duration is 3 min, and the water temperature is controlled at 22℃.

[0013] Preferably, in step S1, the female parent is F6 generation of Megalobrama amblycephala "Pujiang No. 2", and the male parent is F3 generation of Culter alburnus from Dianshanhu.

[0014] Preferably, in step S1, the oxytocin drugs include human chorionic gonadotropin (HCG) and luteinizing hormone-releasing hormone A2 (A2 LHRH-A2). Further, the dose of the oxytocin drugs of the female parent is 8-12 μg of human chorionic gonadotropin mixed with 180-220 units of luteinizing hormone-releasing hormone A2 / kg, and the dose of the oxytocin drugs of the male parent is halved, because the male fish is more sensitive to oxytocin than the female fish, and the male fish may be over-excited and agitated after injection of oxytocin, resulting in discomfort symptoms or even death. In addition, halving the dose can make the spermiation of the male fish and the ovulation of the female fish tend to be synchronized, so as to avoid the male fish from spermiation in advance.

[0015] Preferably, in step S1, after the artificial insemination is completed, talcum powder is used for desmoplastic treatment.

[0016] The hydrostatic pressure method is a kind of triploid induction method that changes the original microtubule structure in the fertilized egg by high-strength pressure, makes the spindle unable to form, temporarily blocks the meiosis process, inhibits the discharge of the second polar body of the fertilized egg, and doubles the chromosome number. Compared with other existing methods for artificially inducing fish triploids, the hydrostatic pressure method has a more standardized procedure and less damage to the fertilized egg. Based on this, the hydrostatic pressure method is selected for the induction of sterile triploids in the hybridization of Misgurnus anguillicaudatus and Culter alburnus. However, the hydrostatic pressure method has obvious application difficulties, that is, the treatment conditions vary with species, and the biological characteristics of the development of the fertilized eggs of different species are different; even for the same species, the maturity of the gametes or the quality difference of the gametes will affect the induction of triploids. At present, the proportion and survival rate of triploids induced by the hydrostatic pressure method are quite different in different fish species: the proportion of triploids induced in cold-water fish such as salmon and trout, which produce thick outer membrane and sink eggs, can reach 90%, and the survival rate can reach 80%, which has been successfully used in commercial production; but in warm-water fish and Cyprinidae fish which produce buoyant or sticky, thin egg membrane and small egg diameter, the efficiency of triploid induction is relatively low, and there is no commercial implementation of triploid production of these fish.

[0017] Misgurnus anguillicaudatus and Culter alburnus belong to the same family of Cyprinidae, and the induction of triploids also has the problem of low efficiency, especially after far-cross hybridization, the induction of triploids is more difficult, and at present, the hydrostatic pressure induction of triploids in the hybridization of Misgurnus anguillicaudatus and Culter alburnus is still blank. Under such technical background, the present application explores different hydrostatic pressure induction conditions (treatment pressure, pressure duration and induction starting time after fertilization), optimizes each related factor, establishes the sterile triploid induction technology of Misgurnus anguillicaudatus and Culter alburnus, and carries out growth performance evaluation test, which confirms the superior growth performance of Misgurnus anguillicaudatus and Culter alburnus triploids, so as to make the batch production and application become a reality.

[0018] Specifically, the present application selects Megalobrama amblycephala as the female parent and Culter alburnus as the male parent, and controls the hydrostatic pressure treatment condition at 2-3 min of the starting time after fertilization, 35-50 MPa of the pressure, and 3 min of the duration, to realize the induction of the Megalobrama amblycephala-Culter alburnus triploid, with the relative hatching rate (i.e. the survival rate) of more than 60%, the triploid rate of more than 50%, and the comprehensive index of more than 75%. When the starting time after fertilization is 3 min, the pressure is 35 MPa, and the duration is 3 min, the highest relative hatching rate of 89.32% can be obtained, but the triploid rate is relatively low (51.36%); when the starting time after fertilization is 2.5 min, the pressure is 50 MPa, and the duration is 3 min, the highest triploid rate of 100% can be obtained, but the relative hatching rate is relatively low (66.5%). The change trend and the change range of the relative hatching rate and the triploid rate are different with the change of the treatment condition, and it is difficult to unify the evaluation standard for the independent evaluation of the indexes, therefore, the present application balances the two indexes by using the comprehensive index to comprehensively evaluate the induction effect, and improves the scientificity, so that the optimal hydrostatic pressure treatment condition is obtained: the starting time after fertilization is 2.5 min, the pressure is 45 MPa, and the duration is 3 min, at this time, the relative hatching rate is 80.06%, the triploid rate is 98.5%, and the comprehensive index reaches the maximum of 87.44%.

[0019] According to the parent selection and the hydrostatic pressure condition control in the method of the present application, the fertilization, hatching and cultivation processes are completed, which can not only realize the induction of the Megalobrama amblycephala-Culter alburnus triploid with high triploidization rate and high survival rate, but also the triploid produced by the induction has obvious advantages in the weight gain rate, the foregut tissue characteristics and the digestive enzyme activity, and has superior growth performance. It is known that the hydrostatic pressure triploids of other fish species including Cyprinidae (Megalobrama amblycephala, black carp, grass carp, silver carp, bighead carp, common carp, crucian carp, etc.) do not have such significant growth promotion, for example, Byamungu et al. found that the growth rate of the triploid juvenile of Oreochromis aureus is not as good as that of the diploid, and Hussain et al. studied the growth of the triploid and the diploid of O. niloticus, and found that there is no significant growth difference between them. Therefore, the significant growth promotion of the present application is an unexpected technical effect, and reflects the superiority of the hydrostatic pressure induction method of the present application.

[0020] It is emphasized that the parent selection of the present application is also crucial, and only when the female parent is Megalobrama amblycephala and the male parent is Culter alburnus, good induction effect (high hatching rate, high triploid rate and superior growth performance) can be obtained. The hybridization combination of Megalobrama amblycephala and Culter alburnus is inter-subfamily hybridization, and the survival rate is very low in inter-species and inter-generic hybridization above the species, and the survival rate is even lower after hydrostatic pressure treatment. However, the survival rate of the inter-subfamily hybridization of Megalobrama amblycephala and Culter alburnus is very high, and the survival rate and triploidization rate of the hybridization of their hybridization eggs after hydrostatic pressure induction are very high, which is a very unexpected discovery, breaks the convention, and is the key basis for the success of the induction method of the present application.

[0021] The second purpose of the present application is to provide the application of the artificial induction method of the above-mentioned Megalobrama amblycephala and Culter alburnus hybrid triploid in fish genetic breeding. The induction method provided by the present application provides a reference for fish distant hybridization triploidization, makes the batch production and application of Megalobrama amblycephala and Culter alburnus triploid fish become a reality, speeds up the breeding process of Megalobrama amblycephala and Culter alburnus excellent line, and promotes the development of Megalobrama amblycephala and Culter alburnus industry.

[0022] The present application has the following beneficial effects:

[0023] 1. The present application successfully induces Megalobrama amblycephala and Culter alburnus hybrid sterile triploid by using hydrostatic pressure method, the triploid rate is high (can reach more than 98%), the survival rate is high (can reach more than 80%), and the comprehensive index is high (can reach more than 85%), which can realize commercial breeding and fill the research blank of Megalobrama amblycephala and Culter alburnus hybrid sterile triploid at present.

[0024] 2. The present application calculates the comprehensive index by taking 60% of the relative hatching rate and 40% of the triploid rate as the weight, comprehensively evaluates the induction effect by introducing the comprehensive index, is more scientific and reasonable, and has more guiding significance.

[0025] 3. The Megalobrama amblycephala and Culter alburnus hybrid sterile triploid induced by the method of the present application has more superior growth performance compared with diploid, and has more breeding potential. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : The relative hatching rate (a), triploid induction rate (b) and comprehensive index (c) of the experimental group under different treatment conditions in Example 1.

[0027] Figure 2 : The relative DNA content of Megalobrama amblycephala diploid (A) and Megalobrama amblycephala triploid (B) in Example 1.

[0028] Figure 3 : The metaphase division phase of chromosomes of Megalobrama amblycephala diploid (left) and Megalobrama amblycephala triploid (right) in Example 1.

[0029] Figure 4Figure 1 shows the karyotype analysis of the control diploid (a) and the experimental triploid (b) in Example 1.

[0030] Figure 5 Figure 2 shows the morphology of the triploid (SBT), the diploid (EBT) and the offspring of the self-crossing of the parents in Example 2.

[0031] Figure 6 Figure 3 shows the morphology cluster tree of the triploid (SBT), the diploid (EBT), the common carp (Cyprinus carpio) and the topmouth gudgeon (Pseudorasbora parva) in Example 2.

[0032] Figure 7 Figure 4 shows the paraffin sections of the foregut tissue of the triploid (a, c) and the diploid (b, d) in Example 3. a, b and c, d are HE staining and AB-PAS staining, respectively. SM: submucosa, S: serosa, wF: fold width, hF: fold height, MS: muscularis, S: mucosa, I: type I mucous cells, II: type II mucous cells, III: type III mucous cells, IV: type IV mucous cells. DETAILED DESCRIPTION

[0033] The present application is further described below in conjunction with the accompanying drawings and specific examples.

[0034] Example 1: Establishment of the artificial induction method of the triploid hybrid of the common carp (Cyprinus carpio) and the topmouth gudgeon (Pseudorasbora parva)

[0035] 1.1 Parental induction, artificial insemination and hatching

[0036] In mid-May, the static water pressure induction experiment of the hybrid of the common carp (♀) and the topmouth gudgeon (♂) was carried out. The female parent was the F6 generation of the common carp "Pujiang No. 2" from the Agricultural Ministry of the People's Republic of China Common Carp Genetic and Breeding Center, and the male parent was the F3 generation of the topmouth gudgeon in Dianshan Lake. Both parents were selected for the breeding experiment, with good gonadal development, strong body, and beautiful type and body of 3-year-old fish (strong static water pressure tolerance). The induction drug (10 μg HCG mixed with 200 units of LHRH-A2 / kg) was injected in the evening, and the male fish was halved in dose. The parent fish was temporarily raised in a circular spawning pool (about 2 m in diameter and about 1.3 m in depth), and the flow water was turned on at the same time. The next morning, when the parents could squeeze out the eggs and sperm, the parent fish was quickly caught, and the eggs and sperm were squeezed out of the parent fish's abdomen in a basin in a light-proof environment for artificial dry insemination, and then talcum powder was used for desmopexy treatment. The fertilized eggs were treated with static water pressure using a static water pressure machine, and then hatched in a hatching barrel until the fry could swim normally, and then transferred to a 1.5-meter-deep 600m 2 large earth pond. The water temperature was controlled at 22-24°C throughout the process from fertilization, hatching to fry cultivation.

[0037] 1.2 Hydrostatic pressure induction condition exploration

[0038] To explore the best conditions for hydrostatic pressure induction of triploid crucian carp, the starting time of post-fertilization treatment was set at 2, 2.5, and 3 minutes, the pressure was set at 35, 40, 45, and 50 MPa, and the treatment duration was 3 minutes at a water temperature of 22℃. The specific experimental design is shown in Table 1 below.

[0039] Table 1 Induction conditions of experimental groups (3x4x1)

[0040]

[0041] According to the method in 1.1, artificial induction was performed, and the induction effects under different hydrostatic pressure induction conditions were evaluated by calculating the relative hatching rate, triploid rate, and comprehensive index. The specific method is as follows:

[0042] Calculation of relative hatching rate: 2000 fertilized eggs from each experimental group and the control group (fertilized eggs without treatment, directly placed in the hatching barrel for hatching, as a blank control) were placed in different labeled hatching barrels for hatching under the same conditions. The number of eggs hatched was counted to calculate the hatching rate, and the relative hatching rate = experimental group hatching rate / control group hatching rate.

[0043] Calculation of triploid rate: Under the same cultivation conditions, the ploidy of 200 randomly selected juvenile fish from each experimental group was detected when they reached 2-5g. Using standard diploid crucian carp fish blood as a reference, 1uL of blood was taken from the caudal vein using a 1mL sterile syringe soaked in heparin sodium solution. After staining for 30s in 800uL of DAPI staining solution, the relative DNA content of blood cells was measured and the ploidy was determined using a ploidy analyzer. The triploid rate = number of triploids detected / total number of samples detected.

[0044] Calculation of comprehensive index: The comprehensive index was calculated using the relative hatching rate of 60% and the triploid rate of 40% as weights (reference Zhang Xin-hui. Induction of triploid and gynogenesis of mandarin fish and study on its main biological characteristics [D]. Wuhan: Huazhong Agricultural University, 2013).

[0045] The relative hatching rate, triploid rate, and comprehensive index of triploid crucian carp under different treatment conditions are shown in Table 2 and Figure 1It can be seen that the relative hatching rate of the triploid experimental group of Pseudobagrus eupogus was 62.23-89.32%, and the triploid rate was 51.36-100%. The starting time and pressure size of post-fertilization treatment had significant effects on the relative hatching rate and triploid rate of Pseudobagrus eupogus triploids. Within a certain pressure range, the relative hatching rate of triploids basically decreased significantly with the increase of pressure (P<0.05), while the triploid rate increased significantly with the increase of pressure (P<0.05). In summary, when the water temperature was 22℃, the starting time of post-fertilization treatment was 3min, the pressure was 35MPa, and the duration was 3min, the highest relative hatching rate (89.32%) could be obtained; when the starting time of post-fertilization treatment was 2.5min, the pressure was 50MPa, and the duration was 3min, the highest triploid rate (100%) could be obtained; when the starting time of post-fertilization treatment was 2.5min, the pressure was 45MPa, and the duration was 3min, the maximum comprehensive index (87.44%) could be obtained, and the comprehensive induction effect was the best.

[0046] Table 2 Relative hatching rate, triploid rate and comprehensive index of experimental groups under different treatment conditions

[0047]

[0048] 1.3 Triploid identification

[0049] The DNA content of the sample fish was detected using a ploidy analyzer, and the results are shown in Table 2. Figure 2 The relative DNA content of the diploid was about 50, which was consistent with the standard diploid crucian carp used for comparison, and the relative DNA content of the triploid was about 75, which was 1.5 times that of the control group. Thus, the induced successful Pseudobagrus eupogus triploids and the uninduced successful Pseudobagrus eupogus diploids were screened out.

[0050] The chromosomes of the Pseudobagrus eupogus triploids and diploids identified by the ploidy analyzer were prepared, and the metaphase of all chromosomes was selected, carefully observed and counted to determine the number of chromosomes, and the metaphase with higher quality was selected for photography Figure 3 ), and then the chromosome karyotype analysis Figure 4 ) was performed by PS 2019 software. It can be seen that the Pseudobagrus eupogus diploid contains 48 chromosomes, the karyotype is 2n=14m+28sm+6st, and the arm number is 90; the chromosome number of the Pseudobagrus eupogus triploid is 72, the karyotype is 3n=21m+42sm+9st, and the arm number is 135; the chromosome number of the Pseudobagrus eupogus triploid is 1.5 times that of the diploid, and one set of chromosomes more than the diploid; a group of the largest chromosomes was observed in the metaphase of both fish, which was judged as sm type by karyotype analysis. The successful induction of Pseudobagrus eupogus triploids was verified by chromosome preparation technology and karyotype analysis.

[0051] Example 2 Morphological difference analysis

[0052] 2.1 Morphological characteristics

[0053] The appearance of the self-crossed offspring of blunt snout bream (TTF), the self-crossed offspring of topmouth bream (QZB), and the triploid bream (SBT) and diploid bream (EBT) obtained by hydrostatic hybridization in Example 1 are as follows. Figure 5 As shown. Careful observation reveals that the triploid's morphological characteristics are quite similar to the diploid's, with most traits falling between their parents. The triploid is taller than the diploid but shorter, similar to the maternal TTF. The triploid's back and upper sides are bluish-gray, while the abdomen is silvery-white, slightly resembling the paternal QZB. Its scales are larger, more like the maternal, but thinner and softer, more like the paternal. The diploid is similar, visually indistinguishable from the triploid. Furthermore, the heads of both the diploid and triploid are very similar, significantly larger than the offspring of both parents, and both have upturned mouths, though less pronounced than the paternal.

[0054] 2.2 Cluster Analysis

[0055] Euclidean distance was used to perform shortest-distance hierarchical clustering on nine traditional measurable proportionate traits and 20 corrected frame structure data (the frame structure measurement method is referenced from Chen Jie, Li Fugui. Morphological differences among different bream populations [J]. Journal of Shanghai Ocean University, 2014, 23(3):388-394.). The morphological distances of the four populations are shown in Table 3 below. The Euclidean distance between the triploid and diploid bream was 0.534, the Euclidean distance with the maternal blunt-snout bream was 1.078, and the Euclidean distance with the paternal topmouth bream was 2.290; the Euclidean distance between the diploid and the maternal blunt-snout bream was 1.504, and the Euclidean distance with the paternal topmouth bream was 2.168; indicating that the triploid is closer to the maternal parent than the diploid in terms of measurable traits and frame structure. The clustering results are as follows. Figure 6 As shown, triploids first clustered with diploids into one group, then with the maternal parent *Brucea buxiflora*, and finally with the paternal parent *Culter alburnus*. This indicates that triploids and diploids are more influenced by the maternal parent than the paternal parent in terms of measurable traits and framework structure.

[0056] Table 3. Euclidean distances of triploid, diploid, blunt-snout bream, and topmouth culter.

[0057]

[0058] Example 3 Growth Performance Evaluation

[0059] 3.1 Weight gain rate

[0060] The growth comparison test of the triploid, diploid and their parents for 210 days is shown in Table 4. The absolute weight gain rate of the triploid is significantly higher than that of other groups. Compared with the diploid, the triploid increases by 8.6%, compared with the self-crossed offspring of the Amur catfish, the triploid increases by 20.5%, and compared with the self-crossed offspring of the Topmouth culter, the triploid increases by 1.2 times, which has a faster growth rate (0.88±0.11 g / d).

[0061] Table 4 Comparison of growth rates of triploid, diploid, Amur catfish and Topmouth culter

[0062]

[0063] Note: The same row with different letters indicates significant difference (P<0.05), the same below.

[0064] 3.2 Histological characteristics of the foregut

[0065] The paraffin sections of the triploid and diploid foregut tissues are treated with HE staining and AB-PAS staining, and the results are shown in Figure 7 From the figure, it can be clearly seen that the intestinal tract has mucosa layer, submucosa layer, muscle layer and serosa layer, and the boundaries of each layer are obvious. Further measurement and analysis of the tissue morphology show that the triploid has a significantly higher wrinkle height, wrinkle width and muscle layer thickness than the diploid (see Table 5 below), indicating that the triploid has a significantly stronger absorption capacity of intestinal nutrient substances than the diploid.

[0066] AB-PAS staining can divide the mucus cells into four types according to their coloring characteristics: type I red, type II blue, type III purple red and type IV blue purple; among them, type I and type III mainly contain neutral mucopolysaccharides that make PAS positive, and type II and type IV mainly contain acid mucopolysaccharides that make AB positive; neutral mucopolysaccharides can form a mucus film on the surface at the mucosa to effectively avoid mechanical damage and pathogen invasion of the intestinal tract, and acid mucopolysaccharides can lubricate food and also form a complex with protease to stabilize the enzyme. Figure 7 As can be seen from the figure, the triploid foregut contains a large number of type I mucus cells dyed red, while such cells do not appear much in the diploid, suggesting that the triploid may have stronger immune capacity than the diploid; in summary, the total number of mucus cells in the triploid is significantly more than that in the diploid, and it can be seen that the triploid has stronger digestive capacity.

[0067] Table 5 Histological characteristics of the foregut

[0068]

[0069] 3.3 Digestive enzyme activity

[0070] Accurately take 0.1 g of foregut tissue of each fish, add 0.9 ml of 0.7% physiological saline to prepare a homogenate under ice water bath condition, centrifuge on a centrifuge for 10 minutes (4°C, 2500 r / min), take the supernatant for detection, and the whole process is operated on ice. The concentration of tissue protein in the supernatant is determined by Coomassie brilliant blue method, the activities of each digestive enzyme are determined according to the steps in the instruction manual of Nanjing Jiancheng, and the absorbance value is measured by an enzyme marker, and the activities of each digestive enzyme in the sample are calculated by using the calculation formula in the instruction manual.

[0071] The results of the enzyme activity determination of the foregut tissue of the triploid and diploid of Culter oxycephalus are shown in Table 6. It can be seen that in the foregut tissue, the trypsin activity of the two fish species is the highest, the amylase activity is the second, and the lipase activity is the lowest, which shows that the intestines of the triploid and diploid of Culter oxycephalus have strong protein absorption capacity. The triploid and the diploid have significant differences in the activities of trypsin, lipase and amylase (P<0.05), and the triploid is significantly lower than the diploid in the activity of lipase (P<0.05), and the rest is significantly greater than the diploid. From the fact that the triploid is greater than the diploid in the activity of amylase and lower than the diploid in the activity of lipase, it is possible that it is related to the diet, and it is speculated that the diet of the triploid may be more similar to that of the Cyprinus carpio haematopterus, which is omnivorous and slightly herbivorous.

[0072] Table 6 Digestive enzyme activity of 10% foregut tissue

[0073]

[0074] Comparative example

[0075] In Comparative Examples 1-3, different combinations of the male parent and the female parent were tried to induce triploidy, and the induction method was the same as that in Example 1 (optimal hydrostatic pressure condition was used). The specific parent selection and induction results are shown in Table 7. It can be seen that the parent combinations of Comparative Examples 1-3 cannot obtain good induction effect; only when the female parent is Cyprinus carpio haematopterus and the male parent is Culter oxycephalus, high hatching rate, high triploid rate and superior growth performance can be obtained, and the successful application of hydrostatic pressure induction to the triploid of the hybrid of Culter oxycephalus can be realized.

[0076] Table 7 Triploid induction test under different parent combinations

[0077]

[0078] Note: The Cyprinus carpio haematopterus and Culter oxycephalus used in Comparative Examples 1-3 are the same as those in Example 1; the specific comparative data of the growth performance are shown in Example 3.

[0079] This specific embodiment is only an explanation of the present application, and is not a limitation of the present application. Any change made by a person skilled in the art after reading the specification of the present application will be protected by the patent law as long as it is within the scope of the claims of the present application.

Claims

1. A method for artificially inducing triploid hybrids of Megalobrama amblycephala and Culter alburnus, characterized in that: The method comprises the following steps: S1. Injecting the fish with a labor-inducing drug, and then placing the fish in an egg-laying pool with running water. When the fish can squeeze out eggs and sperm, the eggs and sperm are squeezed out from the abdomen of the fish under light-proof environment for artificial insemination; S2. Treating the fertilized eggs with hydrostatic pressure, and the treatment conditions are as follows: the treatment is started 2.5 minutes after fertilization, the pressure is 45 MPa, the duration is 3 minutes, and the water temperature is controlled at 22°C; S3. After the hydrostatic pressure treatment, the fertilized eggs are hatched in a hatching barrel until the fry can swim normally, and then the fry are transferred to a large soil pond for breeding. The water temperature during the whole process of fertilization, hydrostatic pressure treatment, and hatching to fry breeding is controlled at 22-24°C. The DNA content is determined by a ploidy analyzer to determine the ploidy of the hybrid fish, and the triploid hybrid of Megalobrama amblycephala and Culter alburnus is obtained. The induction effect is evaluated by a comprehensive index.

2. The artificial induction method of triploid hybrids of Megalobrama amblycephala and Procyprinus summerae according to claim 1, characterized in that: In step S1, the female parent is the F6 generation of Megalobrama amblycephala "Pujiang No. 2", and the male parent is the F3 generation of Culter alburnus from Dianshui Lake.

3. The artificial induction method of triploid hybrids of Megalobrama amblycephala and Procyprinus summerae according to claim 1, characterized in that: In step S1, the labor-inducing drug comprises human chorionic gonadotropin and luteinizing hormone-releasing hormone A2.

4. The artificial induction method of triploid of hybrid between Megalobrama amblycephala and Procyprinus summerae according to claim 3, characterized in that: The dose of the labor-inducing drug for the female parent is 8-12 μg of human chorionic gonadotropin mixed with 180-220 units of luteinizing hormone-releasing hormone A2 / kg, and the dose of the labor-inducing drug for the male parent is halved.

5. The artificial induction method of triploid of hybrid between Megalobrama amblycephala and Procyprinus summerae according to claim 1, characterized in that: In step S1, talcum powder is used for desmoplastic treatment after artificial insemination.

6. The application of the artificial induction method of the triploid hybrid of Megalobrama amblycephala and Culter alburnus according to any one of claims 1-5 in fish genetic breeding.

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