Method for improving artificial breeding efficiency of oyster in Hong Kong, China by using riboflavin and application thereof

Abstract

The present application relates to the field of aquaculture technology, in particular to a method for improving the efficiency of artificial breeding of Hong Kong oyster and application, the present application adopts two ways of treating Hong Kong oyster gametes or parent oysters with riboflavin, which can significantly improve the efficiency of artificial breeding of Hong Kong oyster, and the hatching efficiency of artificial embryos of Hong Kong oyster is significantly improved; through experiments, when the parent oysters of Hong Kong oyster are treated with riboflavin, the final concentration of riboflavin is 3mg / L, the salinity of seawater is 12‰, the hatching rate of oysters is increased from 16.25% of the control group to 82.99%, which is increased by 4.11 times; when the gametes of Hong Kong oyster are treated with riboflavin, the sperm and egg of Hong Kong oyster need to be mixed and soaked in 1mg / L riboflavin seawater solution, and when the salinity of seawater is 12‰, the hatching rate of oysters is increased from 49.78% of the control group to 76.48%, which is increased by 53.64%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquaculture, in particular to a method for improving the efficiency of artificial breeding of Crassostrea hongkongensis and application thereof. BACKGROUND

[0002] Crassostrea hongkongensis belongs to the genus Crassostrea, and the shell color is mostly green. It mainly inhabits in the coastal waters of southern China and Vietnam. It is commercially cultured due to its large size, high nutritional value, and rapid growth. Currently, due to climate and environmental changes, the natural seed yield of Crassostrea hongkongensis is unstable, and the quantity and quality cannot meet market demand. During the breeding process, season and environmental factors such as temperature, salinity, and available phytoplankton all affect the growth of Crassostrea hongkongensis. Oyster death is also related to many factors such as temperature, stress, sexual maturity, pathogens, and pollutants. Currently, Crassostrea hongkongensis mainly exhibits problems such as natural discharge barrier of male and female gametes, low fertilization rate, low hatching rate, high malformation rate, and dramatic reduction in the number of D-type larvae, which further leads to degradation of germplasm resources.

[0003] In artificial breeding production practice, in order to solve the problem of natural discharge barrier of male and female gametes of Crassostrea hongkongensis, the method of dissecting and collecting sperm and eggs for artificial insemination and hatching is usually used. Although this method solves the discharge barrier problem, the maturity of the sperm and eggs obtained by dissection is inconsistent, which still leads to low fertilization rate, low hatching rate, high malformation rate, and low number of hatched D-type larvae, resulting in low hatching efficiency.

[0004] In the prior art, in northern China, the method of temperature rise and nutrition enhancement is mainly used to promote the maturation of oyster gonads, and good hatching effect has been achieved. However, Crassostrea hongkongensis grown in Guangdong and Guangxi has been living in high water temperature for a long time, that is, the biological zero is higher, and the temperature rise range is limited, so the effect of temperature rise for maturation is poor. Nutrition enhancement also needs to be carried out in low water temperature to achieve better effect, which is not suitable in the case of high water temperature in South China. The main reasons are: first, the water temperature in Guangdong and Guangxi is high, and Crassostrea hongkongensis has fast metabolism, so it is not easy to accumulate nutrition and convert it into reproductive performance. Second, if some high-nutrition substances such as egg yolk, starch, yeast, and Schizochytrium are put in high water temperature, the water quality will deteriorate immediately, which not only does not achieve the effect of nutrition enhancement, but also causes a large number of deaths.

[0005] Riboflavin (VB2) is a water-soluble B vitamin, which is the precursor of many flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) coenzymes, which are widely involved in sugar metabolism, fatty acid beta oxidation, protein metabolism, respiratory chain electron transport and redox reaction system and other metabolic processes. The prior art reports that feeding riboflavin to egg-laying animals such as chickens and ducks can make the hens transfer riboflavin in the body to the eggs, thereby reducing the mortality of the embryos. However, as a kind of mollusk, the reproductive process of oysters is quite different from that of egg-laying animals. Whether riboflavin can be used to improve the efficiency of artificial breeding of oysters is a problem that has not been studied in the current research field. Therefore, in the reproductive process of oysters, how to find a suitable promoter for the reproduction of reproductive cells of Hong Kong oysters in the southern water area and improve the reproductive performance of oysters in combination with the characteristics of the water area is an effective solution to improve the quality of oyster larvae. SUMMARY

[0006] In view of the above, in order to overcome the shortcomings of the prior art, the present application provides a new method for improving the efficiency of artificial breeding of Hong Kong oysters in a short period of time using riboflavin. The method finds a suitable promoter for the reproduction of reproductive cells of Hong Kong oysters in the southern water area and effectively improves the efficiency of artificial breeding of oysters in combination with the characteristics of the water area, thereby greatly improving the hatching efficiency.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] The application of riboflavin in improving the efficiency of artificial breeding of Hong Kong oysters, wherein the concentration of riboflavin is 1mg / L-3mg / L.

[0009] The present application also includes a method for improving the efficiency of artificial breeding of Hong Kong oysters using riboflavin. The method for improving the efficiency of artificial breeding of Hong Kong oysters using riboflavin is characterized in that the method comprises treating Hong Kong oyster parents or treating Hong Kong oyster gametes with riboflavin; the concentration of riboflavin is 1mg / L-3mg / L; the salinity of seawater for treating Hong Kong oyster parents is 12‰; and the Hong Kong oyster gametes are treated by mixing sperm and eggs and then putting them into 1mg / L riboflavin seawater solution.

[0010] Further, the method for treating Hong Kong oyster parents comprises the following specific steps:

[0011] S1, parent oyster temporary cultivation: temporarily cultivating Hong Kong oyster parents in the breeding period in a cultivation pond;

[0012] S2, preparation before treatment: preparing three cement pools, adjusting the salinity of seawater in the pools to 12‰, and adding riboflavin in the first cement pool in advance and adjusting the concentration to 3mg / L;

[0013] S3, riboflavin treatment: the Chinese Hong Kong oyster parent oysters in step S1 are put into the first cement pool in step S2 for treatment for 24 hours; after 24 hours, they are transferred to the second cement pool, and the riboflavin concentration in the pool is 3 mg / L, and the treatment is continued for 24 hours; after 24 hours, they are transferred to the third cement pool, and the riboflavin concentration in the pool is 3 mg / L, and the treatment is continued for 24 hours; the total treatment time is 72 hours; the treatment density of the Chinese Hong Kong oyster parent oysters is 100-150 kg / m 3 ,

[0014] S4, artificial breeding: after the treated Chinese Hong Kong oyster parent oysters are dissected and the male and female are distinguished, the traditional method is adopted to perform artificial insemination and hatching, and the seawater salinity during hatching is 12 ‰.

[0015] Further, the seawater temperature in the S2 step is 25-28°C.

[0016] Further, the method for treating the gametes of the Chinese Hong Kong oyster comprises the following specific steps:

[0017] ①Obtaining sperm and eggs of the Chinese Hong Kong oyster: after the Chinese Hong Kong oyster parent oysters that have been temporarily cultured indoors for 3-15 days and are in the breeding period, the male and female are distinguished, and the sperm and eggs are obtained by dissection, respectively;

[0018] ②Riboflavin treatment: after the sperm and eggs are mixed and left for 10 minutes after being quantitatively treated, respectively, the sperm and egg mixture is transferred to a container containing a riboflavin treatment solution with a salinity of 12 ‰, and the final concentration of riboflavin in the container is 1 mg / L, and the treatment time is 15 minutes;

[0019] ③Elution: after 15 minutes of treatment in step ②, the sperm and egg mixture in step ② is quickly eluted with a 400-mesh silk screen, and the treatment solution is washed away;

[0020] ④Hatching: the sperm and egg mixture eluted in step ③ is placed in seawater with a salinity of 12 ‰ for hatching.

[0021] Further, the method for quantitatively treating the sperm and eggs in step ② is to dilute the sperm and eggs with seawater with a salinity of 12 ‰ and a water temperature of 28°C, respectively, to obtain a sperm mother liquor and an egg mother liquor, wherein the density of the egg mother liquor is 50 pieces / ml, and the total volume is 10 L; the density of the sperm mother liquor is 10 2 -10 3 pieces per microliter, and the total volume is 10 L.

[0022] Further, the mixing ratio of the sperm and eggs in step ② is 5-10:1.

[0023] Further, the hatching temperature in step ④ is 27-30°C.

[0024] The present application has the following advantages:

[0025] 1、The present application significantly improves the efficiency of artificial breeding of Chinese Hong Kong oysters by using riboflavin of appropriate concentration, treating parent oysters of Chinese Hong Kong oysters under appropriate seawater salinity, and treating gametes of Chinese Hong Kong oysters under appropriate methods, which greatly improves the hatching efficiency in the artificial breeding production of Chinese Hong Kong oysters. Experimental data show that when the concentration of riboflavin is 3 mg / L, the parent oysters of Chinese Hong Kong oysters are treated under 12 ‰ seawater salinity, and then artificial insemination and hatching are performed, the artificial breeding efficiency reaches the optimum, among which the fertilization rate reaches 89.18%, which is increased by 91.13% compared with the control group, the hatching rate reaches 82.99%, which is increased by 4.11 times compared with the control group, and the deformity rate of D-type larvae hatched is 3.26%, which is reduced by 93.48% compared with the control group; and in the method of treating Chinese Hong Kong oyster gametes, after the sperm and eggs are mixed, the method of treating with 1 mg / L concentration of riboflavin for 15 minutes, the fertilization rate of Chinese Hong Kong oyster embryos is 82.04%, which is increased by 81.06% compared with the control group, the hatching rate is 76.48%, which is increased by 53.64% compared with the control group, and the deformity rate of D-type larvae hatched is 28.93%, which is reduced by 48.83% compared with the control group. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is a two-factor variance analysis diagram of riboflavin treatment of Chinese Hong Kong oyster parent oysters under different seawater salinity and different concentration gradients; Figure 1 A is a fertilization rate result diagram; Figure 1 B is a hatching rate result diagram; Figure 1 C is a deformity rate result diagram.

[0027] Figure 2 It is a two-factor variance analysis diagram of riboflavin treatment of Chinese Hong Kong oyster reproductive cells under four different treatment methods and different concentration gradients; Figure 2 A is a fertilization rate result diagram; Figure 2 B is a hatching rate result diagram; Figure 2 C is a deformity rate result diagram.

[0028] Figure 3 It is a micrograph of D-type larvae after artificial insemination and hatching under four treatment methods. DETAILED DESCRIPTION

[0029] All features disclosed in this specification, or all steps of any methods or processes disclosed, may be combined in any combination, except combinations where at least some features and / or steps are mutually exclusive.

[0030] Any feature in the foregoing specification that is expressed as a particular combination of features, unless specifically stated otherwise, can be expressed or performed with reference to alternate combinations characterised by one or more features other than those features expressing the particular combination.

[0031] Example 1

[0032] This example studies the effects of different concentrations of riboflavin on the artificial breeding efficiency of Chinese Hong Kong oyster parent oysters under different salinities, including the following steps:

[0033] S1, collect and temporarily cultivate Chinese Hong Kong oyster parent oysters: in May 2022, 240 kg of 3-year-old Chinese Hong Kong oyster parent oysters with a size of 7.83 ± 2.16 cm were collected in Sandun sea area of Guangxi and returned to the hatchery. After cleaning the surface attachments and sludge, they were placed in 12 culture cages in a 20 m cubic meter of water in a cement pool for temporary cultivation for 3 days. The salinity of the temporary seawater was 15%, the water temperature was 28℃, and sufficient chlorella was fed and the air was kept.

[0034] S2, preparation before treatment: considering the economy of drug use, high-density and short-term treatment methods are used, 36 100L treatment containers with fresh filtered seawater are prepared in advance, the water volume is 80L, and they are divided into two groups: high-salinity group and low-salinity group, each group has 18 treatment containers. The salinity of the seawater in the high-salinity group is adjusted to 24‰, and the salinity of the seawater in the low-salinity group is adjusted to 12‰. The water temperature is 30℃, and then riboflavin is added to the first 6 treatment containers in each group, with concentrations of 0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L, respectively. The seawater temperature in this step is maintained at 25-28℃.

[0035] S3, riboflavin treatment: 12 culture cages containing Chinese Hong Kong oyster parent oysters are placed in 6 concentration gradient treatment containers in the high-salinity group and the low-salinity group for the first treatment, and the density of Chinese Hong Kong oyster parent oysters is 100 kg / m 3 After 24 hours of treatment, the second treatment is performed according to the method of S2, riboflavin is added to the other 12 treatment containers in each group, and the final concentration of riboflavin corresponds to the first treatment (0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L, respectively). After 24 hours of treatment, the third treatment is performed according to the method of S2, riboflavin is added to the other 12 treatment containers in each group, and the final concentration of riboflavin corresponds to the first treatment (0 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L, respectively). After 24 hours of treatment, the treatment process is completed, and the total treatment time is 72 hours. No water change is performed during the treatment period, and sufficient chlorella is fed and the air is kept.

[0036] S4, Artificial breeding: After the high-salinity group and low-salinity group and different concentration gradient groups of Chinese Hong Kong oyster parent shellfish were dissected and distinguished between male and female, the traditional method was used for in-group artificial insemination and hatching. The seawater salinity was 12‰ and the seawater temperature was 27-30℃ during hatching. The fertilization rate, hatching rate and deformity rate of the high-salinity group and low-salinity group under different concentration gradients were measured and two-factor variance analysis was performed. The calculation method of fertilization rate, hatching rate and deformity rate was as follows:

[0037] ① Fertilization rate (%) = number of fertilized eggs / total number of eggs x 100;

[0038] ② Hatching rate (%) = number of hatched larvae / number of fertilized eggs x 100;

[0039] ③ Deformity rate (%) = number of deformed larvae / number of hatched larvae x 100.

[0040] This experiment was repeated three times. The statistical results of fertilization rate, hatching rate and deformity rate are shown in Tables 1-2,

[0041] Table 1 Fertilization rate of Chinese Hong Kong oyster after riboflavin treatment under different salinities

[0042]

[0043] Note: The same row and different lowercase letters represent significant difference (P<0.05), and the "*" indicates extremely significant difference (P<0.001).

[0044] From the experimental results in Table 1, under different salinities (12‰ and 24‰), the Chinese Hong Kong oyster parent shellfish treated with riboflavin for 72 hours under six concentration gradients, and then artificial insemination, the seawater salinity and treatment concentration two factors produced different degrees of influence on the fertilization rate. Under the seawater salinity of 24‰, the fertilization rate of the 5mg / L concentration treatment group was the highest, and under the seawater salinity of 12‰, the fertilization rate of the 3-5mg / L concentration treatment group was the highest, and both were significantly higher than the 0mg / L control group (P<0.001).

[0045] Table 2 Hatching rate of Chinese Hong Kong oyster after riboflavin treatment under different salinities

[0046]

[0047] Note: The same row and different lowercase letters represent significant difference (P<0.05), and the "*" indicates extremely significant difference (P<0.001).

[0048] As shown in Table 2, after treating Hong Kong oyster parent oysters with riboflavin at six concentration gradients for 72 hours, followed by artificial insemination and hatching, both seawater salinity and treatment concentration had varying degrees of influence on the hatching rate. At a seawater salinity of 12‰, the hatching rate of all treatment groups was significantly higher than that at 24‰ (P<0.05), with the 3 mg / L treatment group exhibiting the highest hatching rate, which was significantly higher than that of the 0 mg / L control group (P<0.001).

[0049] Table 3. Deformity rate of oysters from Hong Kong, China after riboflavin treatment at different salinities.

[0050]

[0051] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05), and "*" indicates extremely significant differences (P < 0.001).

[0052] As shown in Table 3, after treating Hong Kong oyster parent oysters with riboflavin at six concentration gradients for 72 hours, followed by artificial insemination and hatching, both seawater salinity and treatment concentration had varying degrees of influence on the deformity rate. At a seawater salinity of 12‰, the 3 mg / L treatment group had the lowest deformity rate, which was significantly lower than that of the 0 mg / L control group (P < 0.001).

[0053] A two-way ANOVA was performed on the above experimental results, and the results are as follows: Figure 1 As shown in the graph: white bars represent salinity of 24‰, and black bars represent salinity of 12‰. Figure 1 A represents the fertilization rate of oysters from Hong Kong, China. Figure 1 B represents the hatching rate of oysters in Hong Kong, China. Figure 1 C represents the deformity rate of oysters from Hong Kong, China; as can be seen from the figure, at a seawater salinity of 12‰, the fertilization rate was highest in the riboflavin treatment group with a concentration of 3-5 mg / L. Figure 1 A), significantly higher than the 0 mg / L control group and other concentration groups (P<0.05); the 3 mg / L riboflavin treatment group had the highest hatching rate ( Figure 1 B), significantly higher than the 0 mg / L control group and other concentration groups (P<0.05); the 3 mg / L riboflavin treatment group had the lowest malformation rate ( Figure 1C) was significantly lower than 0 mg / L control group and other concentration groups (P < 0.05). Combined with the analysis of the fertilization rate in Table 2, the hatching rate in Table 3 and the deformity rate in Table 4, the artificial breeding efficiency of the Hong Kong oyster reached the optimum after the gametes of the Hong Kong oyster were treated with 3 mg / L riboflavin in 12‰ seawater for 72 hours, then dissected, artificially inseminated and hatched, in which the fertilization rate reached 89.18%, which was 91.13% higher than that of the control group, the hatching rate reached 82.99%, which was 4.11 times higher than that of the control group, and the deformity rate of the D-type larvae was 3.26%, which was 93.48% lower than that of the control group.

[0054] Example 2:

[0055] The embodiment of the present application provides a method for improving the artificial breeding efficiency of Hong Kong oysters by treating the gametes of Hong Kong oysters with riboflavin, which comprises the following steps:

[0056] ①Obtain the gametes of Hong Kong oysters: in July 2022, collect 20 Hong Kong oyster parent oysters temporarily cultured in Guangxi Maowei Sea aquaculture hatchery, the indoor seawater salinity is 12%, the water temperature is 30℃, and sufficient feed is fed. After 15 days of temporary culture, 20 Hong Kong oyster parent oysters are taken, the male and female are distinguished, the sperm and eggs are obtained by dissection, and the sperm and eggs are quantified, wherein the quantification method is: dilute the sperm and eggs with seawater with a salinity of 12‰ and a water temperature of 28℃, finally obtain sperm mother liquor and egg mother liquor, wherein the density of the egg mother liquor is 50 pieces / ml, and the total volume is 10L; the density of the sperm mother liquor is 10 2 -10 3 pieces / microliter, and the total volume is 10L.

[0057] ②Riboflavin treatment: use 4 different treatment methods and 6 different concentration gradients of riboflavin to treat the gametes of Hong Kong oysters, and the treatment time is 15 minutes.

[0058] ③Elution: after 15 minutes of treatment, quickly elute (wash away the treatment solution) the embryos with a 400-mesh silk screen;

[0059] ④Hatching: place the newly eluted treatment solution into a fresh seawater container with a salinity of 12‰ and a water temperature of 27-30℃ for hatching, and perform trace aeration during the hatching process, and measure the fertilization rate, hatching rate and deformity rate, and perform two-factor variance analysis.

[0060] In step 2, the four different treatment methods are as follows: (1) simultaneous treatment of sperm and eggs for re-fertilization, i.e. the eggs and sperm are treated with different concentration gradients of riboflavin at the same time for 15 minutes, and then artificial insemination is performed. The specific operation is as follows: 12 plastic beakers are prepared, and 6 different concentration gradients of riboflavin are added to 6 plastic beakers in advance and dissolved with fresh seawater, and the volume of seawater in each plastic beaker is quantified to 100 ml, then 400 ml of egg mother liquor is quickly added. The same method is used to treat sperm in the other 18 plastic beakers. After the eggs and sperm are treated for 15 minutes, the eggs are first washed with a 400-mesh silk screen (to remove the treatment solution), and the treated eggs are re-placed in a beaker of fresh seawater and the volume is quantified to 500 ml. Then 3-5 ml of sperm treated with the same difference metabolite at the same concentration is added to the plastic cup containing the treated eggs, and incubation is waited. (2) Only the eggs are treated, i.e. the eggs are treated with different concentration gradients of riboflavin for 15 minutes, and then untreated sperm is added for artificial insemination. The specific operation is as follows: 6 plastic beakers are prepared, and 6 different concentration gradients of riboflavin are added to 6 plastic beakers in advance and dissolved with fresh seawater, and the volume of seawater in each plastic beaker is quantified to 100 ml, then 400 ml of egg mother liquor is quickly added. After 15 minutes, the eggs are first washed with a 400-mesh silk screen (to remove the treatment solution), and the treated eggs are re-placed in a beaker of fresh seawater and the volume is quantified to 500 ml. Then 1-3 ml of untreated sperm mother liquor is quickly added, and incubation is performed. (3) Only the sperm is treated, i.e. the sperm is treated with different concentration gradients of riboflavin for 15 minutes, and then untreated eggs are added for artificial insemination. The specific operation is as follows: 12 plastic beakers are prepared, and 400 ml of untreated egg mother liquor is added to 6 plastic beakers, and the volume is quantified to 500 ml with fresh seawater. 6 different concentration gradients of riboflavin are added to the other 6 plastic beakers in advance and dissolved with fresh seawater, and the volume of seawater in each plastic beaker is quantified to 100 ml, then 400 ml of sperm mother liquor is quickly added. After 15 minutes, 3-5 ml of treated sperm is added to the beaker containing untreated eggs, and incubation is performed. (4) The eggs and sperm are treated after mixing, i.e. the fertilized eggs are treated with different concentration gradients of riboflavin for 15 minutes, and then incubation is performed. The specific operation is as follows: 12 plastic beakers are prepared, and 6 different concentration gradients of riboflavin are added to 6 plastic beakers in advance and dissolved with fresh seawater, and the volume of seawater in each plastic beaker is quantified to 100 ml. 400 ml of untreated egg mother liquor is added to the other 6 plastic beakers, and 1-3 ml of untreated sperm mother liquor is quickly added for artificial insemination. After insemination, the fertilized eggs are added to the corresponding treatment solution, treated in the treatment solution for 15 minutes, washed (the method is the same as above), and then placed in a beaker of fresh seawater for incubation.

[0061] In step 2, the 6 different concentration gradients of riboflavin treatment are: 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L.

[0062] The experiment was repeated three times at three different times (July 16, 2023, July 18, 2023, and July 21, 2023). The final statistical results of fertilization rate, hatching rate, and malformation rate are shown in Tables 4-6.

[0063] Table 4 Effect of different concentrations of riboflavin on fertilization rate of Hong Kong oyster under different reproductive cell treatment methods

[0064]

[0065] Note: The same row "*" indicates a significant difference (P<0.001).

[0066] From the experimental results in Table 4, it can be seen that using four different methods (simultaneous treatment of sperm and eggs, treatment of eggs only, treatment of sperm only, and treatment after insemination), and treating Hong Kong oyster dissection embryos with six different concentrations of riboflavin for 15 minutes before artificial insemination, both the treatment method and the treatment concentration have different degrees of influence on the fertilization rate, but there is no significant difference. Under the method of treating after insemination, the fertilization rate of Hong Kong oyster embryos reaches the optimal, and the fertilization rate of riboflavin concentration of 1 mg / L is better, which is significantly higher than that of the control group (P<0.001).

[0067] Table 5 Effect of different concentrations of riboflavin on hatching rate of Hong Kong oyster under different reproductive cell treatment methods

[0068]

[0069] Note: The same row "*" indicates a significant difference (P<0.001).

[0070] As shown in Table 5, the experimental results revealed that using four different methods (simultaneous sperm and egg treatment followed by insemination, egg treatment only, sperm treatment only, and sperm-egg mixture treatment after insemination), and treating dissected oyster embryos from Hong Kong with six different concentrations of riboflavin for 15 minutes before artificial insemination and hatching, both the treatment method and concentration had varying degrees of influence on the hatching rate. The sperm-egg mixture treatment method resulted in the optimal hatching rate for Hong Kong oyster embryos, with the 1 mg / L concentration group exhibiting the highest hatching rate, significantly higher than the 0 mg control group (P < 0.001).

[0071] Table 6. Effects of different concentrations of riboflavin on oyster deformity rates in Hong Kong, China, under different germ cell treatment methods.

[0072]

[0073] Note: The asterisk (*) in the same row indicates a highly significant difference (P < 0.001).

[0074] As shown in Table 6, the experimental results indicate that using four different methods (simultaneous sperm and egg treatment before fertilization, egg treatment only, sperm treatment only, and sperm-egg mixture treatment after insemination), and treating Hong Kong oyster embryos with six different concentrations of riboflavin for 15 minutes before artificial insemination and hatching, both the treatment method and concentration had varying degrees of influence on the malformation rate. The sperm-egg mixture treatment method resulted in the lowest malformation rate for Hong Kong oyster embryos, with the 1 mg / L and 3 mg / L treatment groups showing the lowest malformation rates, significantly lower than the 0 mg control group (P < 0.001).

[0075] A two-way ANOVA was performed on the above experimental results, and the results are as follows: Figure 2 As shown: The bars in the graph represent different riboflavin concentrations; Figure 2 A, Figure 2 B and Figure 2 The horizontal bar for C: "sperm and eggs" indicates a treatment group where sperm and eggs are treated simultaneously before fertilization; "eggs only" indicates a treatment group where only eggs are treated; "sperm only" indicates a treatment group where only sperm are treated; and "after insemination" indicates a treatment group where sperm and eggs are treated after mixing. Figure 2 As shown in the two-way ANOVA, among the four methods, the fertilization rate of oyster embryos dissected in Hong Kong, China, was the highest under the method of sperm-egg mixing and then processing. Figure 2A), significantly higher than the method of inseminating the ova after treating the gametes (P < 0.05), but not significantly different from the method of treating only the ova and the method of treating only the sperm (P > 0.05); at the same time, the hatching rate was also at a high level Figure 2 B), but not significantly different from the other three methods (P > 0.05); at the same time, the deformity rate was at the lowest level Figure 2 C), but not significantly different from the other three methods (P > 0.05).

[0076] Figure 3 Fig. 4 is a micrograph of the D-type larvae after artificial insemination hatching of the four methods, and the micrographs of the D-type larvae of each method group and concentration group (only the experiment on July 16, 2023 in the three repeated experiments is shown). As can be seen from the figure, in the method of treating after mixing the sperm and the ova, the treatment group of riboflavin at a concentration of 1-3 mg / L has the best hatching effect, mainly manifested as the D-type larvae being more regular in shape and having a low proportion of deformed larvae, which is obviously better than the control group and other concentration groups in the same method. Combined with the analysis of the fertilization rate in Table 5, the hatching rate in Table 6 and the deformity rate in Table 7, after treating the embryos of the Hong Kong oyster with riboflavin at a concentration of 1 mg / L for 15 minutes in the method of treating after mixing the sperm and the ova, and then performing artificial insemination hatching, the artificial breeding efficiency is optimal, in which the fertilization rate is 82.04%, which is increased by 81.06% compared with the control group, the hatching rate is 76.48%, which is increased by 53.64% compared with the control group, and the deformity rate of the D-type larvae hatched is 28.93%, which is reduced by 48.83% compared with the control group.

[0077] In summary, both the two ways of treating the Hong Kong oyster by using riboflavin to treat the parent oysters and the gametes of the Hong Kong oyster can significantly improve the artificial breeding efficiency of the Hong Kong oyster. According to the experiment, when the parent oysters of the Hong Kong oyster are treated with riboflavin, the final concentration of riboflavin is 3 mg / L, and the salinity of seawater is 12‰. It has been verified that the fertilization rate of the Hong Kong oyster treated in this way reaches 89.18%, which is increased by 91.13% compared with the control group, the hatching rate reaches 82.99%, which is increased by 4.11 times compared with the control group, and the deformity rate of the D-type larvae hatched is 3.26%, which is reduced by 93.48% compared with the control group. When the gametes of the Hong Kong oyster are treated with riboflavin, the final concentration of riboflavin is 1 mg / L, and the gametes of the Hong Kong oyster need to be mixed and soaked in the riboflavin seawater solution. The salinity of seawater is 12‰. It has been verified that the fertilization rate of the Hong Kong oyster treated in this way is 82.04%, which is increased by 81.06% compared with the control group, the hatching rate is 76.48%, which is increased by 53.64% compared with the control group, and the deformity rate of the D-type larvae hatched is 28.93%, which is reduced by 48.83% compared with the control group.

[0078] Example 3:

[0079] The same method was used to study the reaction of Ruditapes philippinarum which has similar reproductive mode to the oyster, and the effect of riboflavin on the reproduction of Ruditapes philippinarum was observed, specifically:

[0080] Treatment 1: The optimal conditions of Example 1 were used, that is, the parent oysters of Ruditapes philippinarum were soaked under the condition that the final concentration of riboflavin was 3 mg / L and the salinity of seawater was 12‰, and the treatment method was referred to Example 1. The fertilization rate, hatching rate and deformity rate of Ruditapes philippinarum were detected, and the condition that the salinity of seawater was 12‰ and the concentration of riboflavin was 0 mg / L was used as control group 1.

[0081] Treatment 2: The optimal conditions of Example 2 were used, that is, the sperm and eggs of Ruditapes philippinarum were mixed and then soaked in a solution with a final concentration of 1 mg / L of riboflavin diluted in seawater with a salinity of 12‰, and other treatment methods were referred to Example 1. The sperm and eggs of Ruditapes philippinarum were mixed and then soaked in seawater with a salinity of 12‰ for 15 min as control group 2, and other treatment methods were referred to Example 1.

[0082] The results are shown in Table 7.

[0083] Table 7 Effect of riboflavin on the reproductive performance of Ruditapes philippinarum

[0084]

[0085] Note: Different lowercase letters in the same column represent significant differences (P<0.05).

[0086] As shown in Table 7, under the same treatment conditions, the fertilization rate and hatching rate of Ruditapes philippinarum in both treatment 1 and treatment 2 were significantly lower than those of the control group, and the deformity rate was significantly higher, which indicates that different animals, although having similar reproductive modes, have different effects of riboflavin on their reproductive performance due to different species. In the breeding of Ruditapes philippinarum, riboflavin did not improve the efficiency of artificial breeding of Ruditapes philippinarum.

[0087] In summary, riboflavin has an improving effect on the reproductive performance of Hong Kong oysters, but it may not have the same effect on other animals.

[0088] The above examples only express several embodiments of the present application, and the description is more specific and detailed, but it should not be understood as limiting the scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, some modifications and improvements can be made, which are within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. The use of riboflavin to improve the efficiency of artificial breeding of oysters in Hong Kong, China, characterized in that, The concentration of the riboflavin is 1-3 mg / L.

2. The method of claim 1 for improving the efficiency of artificial breeding of Crassostrea hongkongensis using riboflavin, wherein, The method comprises treating Chinese Hong Kong oyster parent shellfish or gametes with riboflavin; when the Chinese Hong Kong oyster parent shellfish is treated, the concentration of the riboflavin is 1-3 mg / L; the salinity of seawater is 12‰; when the gametes are treated, the sperm and egg are mixed and then soaked in riboflavin.

3. The method of claim 2, wherein, The method for treating the Chinese Hong Kong oyster parent shellfish comprises the following specific steps: S1, temporary cultivation of parent shellfish: the Chinese Hong Kong oyster parent shellfish in the breeding period is temporarily cultivated in a cultivation pond; S2, preparation before treatment: three cement pools are prepared, the salinity of seawater in the pools is adjusted to 12‰, riboflavin is added in the first cement pool in advance, and the concentration is adjusted to 3 mg / L; S3, riboflavin treatment: the Chinese Hong Kong oyster parent shellfish of step S1 is put into the first cement pool of step S2 for treatment for 24 h; after 24 h, it is transferred to the second cement pool, the riboflavin concentration in the pool is 3 mg / L, and the treatment is continued for 24 h; after 24 h, it is transferred to the third cement pool, the riboflavin concentration in the pool is 3 mg / L, and the treatment is continued for 24 h; the total treatment time is 72 h; the Chinese Hong Kong oyster parent shellfish is put at a density of 100-150 kg / m 3 , S4, artificial breeding: after the treated Chinese Hong Kong oyster parent shellfish is dissected and the male and female are distinguished, artificial insemination and hatching are carried out by using the traditional method, and the salinity of seawater during hatching is 12‰.

4. The method of claim 3, wherein, The temperature of seawater in the S2 step is 25-28 ℃.

5. The method of claim 2, wherein, The method for treating the gametes of the Chinese Hong Kong oyster comprises the following specific steps: ①obtaining sperm and egg of the Chinese Hong Kong oyster: after the Chinese Hong Kong oyster parent shellfish that has been temporarily cultivated indoors for 7 days and is in the breeding period, the male and female are distinguished, and the sperm and egg are obtained by dissection respectively; ②riboflavin treatment: the sperm and egg are mixed after being quantitatively separated, and then are placed for 10 minutes, then the mixture is transferred to a container containing riboflavin treatment liquid with a salinity of 12‰, the final concentration of riboflavin in the container is 1 mg / L, and the treatment time is 15 minutes; ③elution: after 15 minutes of treatment in step ②, the mixture of sperm and egg in step ② is eluted with a 400-mesh silk screen, and the treatment liquid is washed away; ④hatching: the mixture of sperm and egg eluted in step ③ is placed in seawater with a salinity of 12‰ for hatching.

6. The method of claim 4, wherein, The method for quantifying the sperm and the egg in step ② is diluting the sperm and the egg respectively with seawater with a salinity of 12 ‰ and a water temperature of 28 ℃, and finally obtaining a sperm mother liquor and an egg mother liquor, wherein the density of the egg mother liquor is 50 eggs / ml, and the total volume is 10 L; the density of the sperm mother liquor is 10 2 -10 3 eggs per microliter, and the total volume is 10 L.

7. The method of claim 4, wherein, The mixing ratio of sperm and egg when mixed in step ② is 5-10:

1.

8. The method of claim 4, wherein, The hatching temperature in step ④ is 27-30 ℃.

Images