A method and application of menthol to enhance the embryonic development ability of Hong Kong oysters

Abstract

This invention relates to the field of aquaculture technology, and particularly to a method and application of menthol in enhancing the embryonic development of *Crassostrea gigas* oysters. The invention employs two methods: treating parent oysters or sperm and eggs with menthol. Both methods significantly enhance the embryonic development of *Crassostrea gigas* oysters. When treating parent oysters with menthol, at a final concentration of 20 mg / L and a seawater salinity of 12‰, the hatching rate increased from 16.25% in the control group to 67.75%, an increase of 3.17 times. When treating gametes with menthol, the sperm and eggs of *Crassostrea gigas* were mixed and soaked in a 20-30 mg / L menthol-infused seawater solution with a seawater salinity of 12‰. The hatching rate increased from 40.85% in the control group to a maximum of 77.06%, an increase of 88.64%.

Description

[Technical Field]

[0001] This invention relates to the field of aquaculture technology, and in particular to a method and application of menthol in enhancing the embryonic development of Hong Kong oysters. [Background Technology]

[0002] Hong Kong oyster (Crassostrea hongkongensis), commonly known as the giant oyster, is an important marine biological resource and economic shellfish in my country, mainly distributed in brackish water areas such as Guangxi and Guangdong. Hong Kong oysters are popular due to their rapid growth, high nutritional value, and strong adaptability to various environments. In Guangxi Zhuang Autonomous Region, Hong Kong oyster farming is mainly concentrated in Qinzhou, Beihai, and Fangchenggang. In recent years, affected by changes in climate and marine environment, the yield of natural Hong Kong oyster seedlings has become unstable, with slow growth rates during offshore cultivation, a high risk of large-scale mortality, and low production efficiency. This has resulted in the quantity and quality of natural seedlings failing to meet market demand, necessitating the urgent need for artificially cultivated Hong Kong oyster seedlings to compensate for the shortage.

[0003] In the artificial breeding and production of Hong Kong oysters, gametes are typically obtained through dissection followed by artificial insemination and hatching. While this method solves the problem of sperm and egg release in Hong Kong oysters, the inconsistent maturity of the sperm and eggs obtained from dissection leads to low fertilization rates, low hatching rates, and high rates of deformities. To address these issues, northern my country primarily uses methods such as heating and nutritional enhancement to promote the maturation of Pacific oyster gonads, achieving good hatching results. However, Hong Kong oysters, which grow in Guangdong and Guangxi provinces, have long lived in higher water temperatures with a higher biological zero point, limiting the extent of temperature increases and resulting in poor maturation effects. Nutritional enhancement is also unsuitable in the higher water temperatures of South China, mainly because the high temperature leads to rapid metabolism in Hong Kong oysters, making it difficult for them to accumulate nutrients and convert them into reproductive performance. Therefore, finding suitable promoters for improving the quality of Hong Kong oyster reproductive cells, specifically tailored to the characteristics of southern waters, and thus enhancing the embryonic development capacity of Hong Kong oysters, is crucial for the artificial breeding industry of Hong Kong oysters.

[0004] Menthol (MEN) has a pungent or sweet taste and is a colorless, needle-shaped or rhomboid white solid. As a common food additive, menthol is often added during food processing to enhance its minty flavor. As a small molecule, menthol interacts with proteins after entering the body through food, further stimulating the organism. Menthol has wide applications not only in the food industry but also in the cosmetics, cigarette, and daily necessities industries. Menthol also has anti-inflammatory properties and is commonly used in the medical field for pain relief, anti-inflammatory and antibacterial purposes. However, there are currently no reports on menthol's ability to improve animal reproductive performance. Due to its anti-inflammatory and antibacterial properties, the applicant unexpectedly discovered that menthol, when used as a bactericide in oyster cultivation, could enhance the developmental ability of oyster embryos. [Summary of the Invention]

[0005] In view of the above, and in view of the shortcomings of the prior art, the present invention provides a novel method for using menthol to enhance the embryonic development ability of Hong Kong oysters in a short period of time. This method is specifically designed for the characteristics of southern waters and effectively enhances the embryonic development ability of Hong Kong oysters, greatly improving the embryonic hatching efficiency.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The application of menthol in enhancing the embryonic development ability of Hong Kong oysters, wherein the concentration of menthol is 20 mg / L-30 mg / L.

[0008] The present invention also includes a method for enhancing the embryonic development ability of Hong Kong oysters using the menthol, the method comprising treating Hong Kong oyster parent stock with menthol or treating Hong Kong oyster gametes with menthol; wherein the concentration of menthol is 20 mg / L-30 mg / L; the salinity of the seawater during the treatment of Hong Kong oyster parent stock is 12‰; and the treatment of Hong Kong oyster gametes is performed by soaking a mixture of sperm and eggs.

[0009] Furthermore, the method for treating Hong Kong oyster parent stock includes the following specific steps:

[0010] S1. Collect and temporarily house Hong Kong oyster parent stock: Collect Hong Kong oyster parent stock in the peak breeding period and temporarily house them in the culture pond;

[0011] S2. Preparation before treatment: Prepare 3 cement tanks filled with fresh filtered seawater in advance, adjust the salinity to 12‰, add menthol to the first cement tank in advance, and adjust the concentration to 20mg / L.

[0012] S3. Menthol Treatment: The Hong Kong oyster broodstock from step S1 were placed in the first cement tank of step S2. The seawater temperature was 27℃-30℃, the salinity was 12‰, and the menthol concentration was 20mg / L. After treatment for 24 hours, they were transferred to the second cement tank, where the seawater temperature, salinity, and menthol concentration were the same as in the first cement tank. Treatment continued for another 24 hours, followed by transfer to the third cement tank for another 24 hours. The stocking density of the Hong Kong oyster broodstock was 200kg / m³. 3 The total processing time is 72 hours;

[0013] S4. Artificial breeding: All the Hong Kong oyster parent oysters treated in S3 were dissected and artificially inseminated using traditional methods. The salinity of the seawater during incubation was 12‰.

[0014] Furthermore, the method for processing the Hong Kong oyster gametes includes the following specific steps:

[0015] ① Obtaining gametes from Hong Kong oysters: Sperm and eggs were obtained from parent Hong Kong oysters that were temporarily cultured in the dissection room for 3-15 days and were in the breeding period;

[0016] ② Menthol treatment: After quantitative analysis of sperm and eggs, mix them and let stand for 10 minutes. Then transfer the sperm and egg mixture to 12‰ seawater with 20-30 mg / L menthol and treat for 15 minutes.

[0017] ③ Washing: After 15 minutes of treatment in step ②, quickly wash the sperm and egg mixture from step ② with a 400-mesh sieve to remove the treatment solution;

[0018] ④ Hatching: The sperm and egg mixture washed out in step ③ is placed in seawater with a salinity of 12‰ for hatching.

[0019] Furthermore, the method for quantifying sperm and eggs in step ② involves diluting the sperm and eggs separately with seawater at a salinity of 12‰ and a temperature of 30℃, ultimately obtaining sperm mother fluid and egg mother fluid. The density of the egg mother fluid is 50 eggs / ml, with a total volume of 10L; the density of the sperm mother fluid is 10... 2 -10 3 Each vial per microliter, with a total volume of 10L.

[0020] Furthermore, in step ②, the mixing ratio of sperm and egg is 5-10:1.

[0021] Furthermore, the incubation temperature in step ④ is 27℃-30℃.

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

[0023] This invention utilizes a suitable concentration of menthol to treat Hong Kong oyster parent stock in a short period of time; or treats Hong Kong oyster gametes using a suitable method. Both methods significantly enhance the embryonic development capacity of Hong Kong oysters and greatly improve hatching efficiency. Experimental data showed that when 20 mg / L menthol was used to treat Hong Kong oyster parent stock at a seawater salinity of 12‰, followed by artificial insemination, the embryonic development capacity was optimal. The fertilization rate reached 96.87%, compared to 46.66% in the control group; the hatching rate reached 67.75%, compared to 16.25% in the control group; and the deformity rate of hatched D-type larvae was 9.87%, compared to 50.00% in the control group. In the method of treating Hong Kong oyster gametes, treating a mixture of sperm and eggs with 20-30 mg / L menthol for 15 minutes before hatching resulted in the highest fertilization rate of 70.10%, an increase of 50.49% compared to the control group; the highest hatching rate reached 77.06%, an increase of 88.64% compared to the control group; and the lowest deformity rate of hatched D-type larvae was 11.39%, a decrease of 88.04% compared to the control group. [Attached Image Description]

[0024] Figure 1 A two-way ANOVA plot of menthol treatment at different concentration gradients in seawater with varying salinity for Hong Kong oyster parents. Figure 1 A shows the fertilization rate results; Figure 1 B shows the hatching rate results; Figure 1 C represents the result graph of the deformity rate.

[0025] Figure 2 A two-way ANOVA plot showing the treatment of Hong Kong oyster germ cells with different concentration gradients of menthol under four different treatment methods.

[0026] Figure 3 Micrographs of D-type larvae after artificial insemination and hatching, processed using four different methods.

Detailed Implementation Methods

[0027] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0028] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0029] Example 1:

[0030] This embodiment studies the effects of different concentrations of menthol on the embryonic developmental capacity of Hong Kong oyster parents under different salinity conditions, including the following steps:

[0031] S1. Collection and temporary rearing of Hong Kong oyster parent stock: In June 2022, 300 kg of 3-year-old Hong Kong oyster parent stock with a size of 8.57±1.32cm were collected from the sea area of ​​Sandun, Guangxi and brought back to the seedling workshop. After cleaning the surface attachments and sludge, they were placed into 12 culture cages and temporarily reared in a culture pond with a volume of 20 cubic meters of water for 3 days. The salinity of the seawater during the temporary rearing was 10-25‰, the water temperature was 27-30℃, and sufficient chlorella was fed and aeration was ensured.

[0032] S2. Preparation before treatment: Considering the economic efficiency of drug use, a high-density, short-term treatment method is adopted. Prepare 36 100L treatment containers filled with fresh filtered seawater in advance, each with a water volume of 80L. Divide them into two groups: a high-salinity group and a low-salinity group, with 18 treatment containers in each group. 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‰. After the water temperature is 30℃, menthol is added to the first 6 treatment containers in each group in advance, and the concentrations are adjusted to 0mg / L, 10mg / L, 20mg / L, 30mg / L, 40mg / L, and 50mg / L, respectively. During this step, the seawater temperature is maintained at 27℃-30℃.

[0033] S3, Menthol Treatment: Twelve culture cages containing Hong Kong oyster broodstock were placed in six treatment containers at six concentration gradients (high-salt and low-salt groups) for the first treatment. The stocking density of the Hong Kong oyster broodstock during treatment was 200 kg / m³. 3 After 24 hours of initial treatment, a second treatment was conducted. Following method S2, menthol was added to the remaining 12 treatment containers in each group, with final concentrations corresponding to those in the first treatment (adjusted to 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively). After 24 hours of initial treatment, a third treatment was conducted. Following method S2, menthol was added to the remaining 12 treatment containers in each group, with final concentrations corresponding to those in the first treatment (adjusted to 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L, respectively). The treatment process ended after 24 hours of initial treatment, with a total treatment time of 72 hours. During the treatment, the water was not changed, and sufficient Chlorella was fed while maintaining aeration.

[0034] S4. Artificial Breeding: After dissecting and identifying the sexes of all parent oysters in the high-salt, low-salt, and different concentration gradient groups, artificial insemination and hatching were performed using traditional methods. The seawater salinity at hatching was 12‰, and the seawater temperature was 27-30℃. The fertilization rate, hatching rate, and deformity rate were measured under different concentration gradients in the high-salt and low-salt groups, and a two-way ANOVA was performed. The calculation methods for fertilization rate, hatching rate, and deformity rate are as follows:

[0035] ① Fertilization rate (%) = Number of fertilized eggs / Total number of eggs × 100;

[0036] ② Hatching rate (%) = Number of larvae hatched / Number of fertilized eggs × 100;

[0037] ③ Deformity rate (%) = Number of deformed larvae / Number of hatched larvae × 100.

[0038] This experiment was repeated three times. The statistical results of fertilization rate, hatching rate, and deformity rate are shown in Tables 1-3.

[0039] Table 1. Fertilization rate of Hong Kong oysters after menthol treatment at different salinities.

[0040]

[0041] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0042] As shown in Table 1, under different salinities (12‰ and 24‰), after treating Hong Kong oyster parent stock with menthol at six concentration gradients for 72 hours and then performing artificial insemination, both seawater salinity and treatment concentration had varying degrees of influence on the fertilization rate. At a seawater salinity of 12‰, the fertilization rate of the 20 mg / L treatment group was the highest, significantly higher than that of the 0 mg / L control group and other concentration groups (P<0.05).

[0043] Table 2 Hatching rate of Hong Kong oysters after menthol treatment at different salinities

[0044]

[0045] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0046] As shown in Table 2, after treating Hong Kong oyster parents with menthol 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 concentration groups was significantly higher than that at 24‰ salinity (P<0.05). The 50 mg / L concentration treatment group had the highest hatching rate, which was significantly higher than that of the 0 mg / L control group and other concentration groups (P<0.05).

[0047] Table 3. Deformity rate of Hong Kong oysters after menthol treatment at different salinities.

[0048]

[0049] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0050] As shown in Table 3, after treating Hong Kong oyster parent stock with menthol 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 20 mg / L concentration treatment group had the lowest deformity rate, significantly lower than the 0 mg / L control group and other concentration groups (P<0.05).

[0051] 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 Hong Kong oysters. Figure 1 B represents the hatching rate of Hong Kong oysters. Figure 1 C represents the deformity rate of Hong Kong oysters; as can be seen from the figure, at a seawater salinity of 12‰, the fertilization rate of Hong Kong oyster parents treated with different concentration gradients of menthol was ( Figure 1 A) Hatching rate ( Figure 1 B) Overall, it was superior to the 24‰ seawater salinity group. At a seawater salinity of 12‰, the fertilization rates were highest in the 20 mg / L and 50 mg / L menthol treatment groups. Figure 1 A), significantly higher than the 0 mg / L control group and other concentration groups (P<0.05); the 50 mg / L menthol 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 20 mg / L menthol treatment group had the lowest malformation rate ( Figure 1 C) was significantly lower than that of the 0 mg / L control group and other concentration groups (P<0.05). Combined with the analysis of fertilization rate in Table 1, hatching rate in Table 2, and malformation rate in Table 3, the optimal embryonic development capacity was achieved when 20 mg / L menthol was used to treat Hong Kong oyster parent stock for 72 hours at a seawater salinity of 12‰, followed by dissection and artificial insemination. The fertilization rate reached 96.87±3.12%, compared to 46.66±2.31% in the control group; the hatching rate reached 67.75±3.95%, compared to 16.25±4.60% in the control group; and the malformation rate of type D larvae was 9.87±0.72%, compared to 50.00±16.67% in the control group.

[0052] Example 2:

[0053] This invention provides a method for enhancing the embryonic development ability of Hong Kong oysters by treating gametes with menthol, comprising the following steps:

[0054] ① Obtaining gametes from Hong Kong oysters: In July 2022, parent Hong Kong oysters were collected from the Maowei Sea area of ​​Guangxi and temporarily raised in the seedling workshop. The indoor seawater salinity was 12%, the water temperature was 30℃, and they were fed sufficient feed. After 15 days of temporary rearing, 20 parent Hong Kong oysters were collected. After identifying the sexes, sperm and eggs were obtained by dissection. The sperm and eggs were then quantified separately. The quantification method was as follows: the sperm and eggs were diluted with seawater with a salinity of 12‰ and a water temperature of 30℃ to obtain sperm mother fluid and egg mother fluid. The density of the egg mother fluid was 50 eggs / ml, and the total volume was 10L; the density of the sperm mother fluid was 10... 2 -10 3 Each vial per microliter, with a total volume of 10L.

[0055] ② Menthol treatment: The gametes of Hong Kong oysters were treated with 4 different treatment methods and 6 different concentration gradients of menthol, with a treatment time of 15 minutes for each.

[0056] ③ Washing: After 15 minutes of treatment, quickly wash the embryos with a 400-mesh sieve (to remove the treatment solution);

[0057] ④ Incubation: The material after the new elution treatment was placed in a container filled with fresh seawater with a salinity of 12‰ and a water temperature of 27-30℃ for incubation. Micro-aeration was carried out during the incubation process, and the fertilization rate, hatching rate and deformity rate were measured and a two-way ANOVA was performed.

[0058] In step ②, the four different treatment methods refer to: (1) Simultaneous treatment of sperm and eggs before insemination, that is, treating sperm and eggs simultaneously with menthol of different concentration gradients for 15 minutes before artificial insemination. The specific operation is as follows: prepare 12 plastic beakers, add 6 different concentration gradients of menthol to 6 plastic beakers in advance and dissolve them with fresh seawater, and measure the volume of seawater in each plastic beaker to 100ml, and then quickly add 400ml of egg mother fluid; treat sperm in the other 18 plastic beakers in the same way; after treating eggs and sperm separately for 15 minutes, first wash the eggs with a 400-mesh sieve (wash away the treatment solution), and put the treated eggs back into the beaker of fresh seawater and measure the volume to 500ml; then Add 3-5 ml of sperm treated with the same differential metabolite at the same concentration to a plastic cup containing the treated eggs, and wait for hatching; (2) Treat only the eggs, that is, treat the eggs with menthol of different concentration gradients for 15 minutes, and then add untreated sperm for artificial insemination. The specific operation is as follows: Prepare 6 plastic beakers, add 6 different concentration gradients of menthol to the 6 plastic beakers in advance and dissolve them with fresh seawater, and measure the volume of seawater in each plastic beaker to 100 ml, and then quickly add 400 ml of egg mother fluid. After 15 minutes, first wash the eggs with a 400-mesh sieve (wash away the treatment solution), put the treated eggs back into the beaker of fresh seawater and measure the volume to 500 ml, and then quickly add 1-3 ml of untreated sperm stock solution and incubate; (3) Treat only the sperm, that is, treat the sperm with menthol of different concentration gradients for 15 minutes, and then add untreated eggs for artificial insemination. The specific operation is as follows: Prepare 12 plastic beakers, add 400 ml of untreated egg stock solution to 6 plastic beakers respectively, and use fresh seawater to measure the volume to 500 ml; add 6 different concentration gradients of menthol to the other 6 plastic beakers in advance and dissolve them with fresh seawater, and measure the volume of seawater in each plastic beaker to 100 ml, and then quickly add 400 ml of sperm stock solution; 15 minutes later, add the corresponding treated sperm to the beaker containing untreated eggs. 3-5 ml of the egg and then incubate; (4) After the sperm and egg are mixed, treat the fertilized eggs with menthol of different concentration gradients for 15 minutes and then incubate. The specific operation is as follows: prepare 12 plastic beakers, add 6 different concentration gradients of menthol to 6 plastic beakers in advance and dissolve them with fresh seawater, and measure the volume of seawater in each plastic beaker as 100 ml; add 400 ml of untreated egg mother fluid to the other 6 plastic beakers and then quickly add 1-3 ml of untreated sperm mother fluid for artificial insemination; after insemination, add the fertilized eggs to the corresponding treatment solution, treat them in the treatment solution for 15 minutes, wash the eggs (the method is the same as above), and add them to the beakers with fresh seawater for incubation.

[0059] In step ②, the six different concentration gradients of menthol treatment are: 0 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, and 50 mg / L.

[0060] This experiment was repeated three times at three different periods (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.

[0061] Table 4. Effects of different concentrations of menthol on fertilization rate of Hong Kong oysters under different germ cell treatment methods.

[0062]

[0063] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0064] As shown in Table 4, the experimental results revealed that using four different methods (simultaneous sperm and egg treatment before fertilization, egg-only treatment, sperm-only treatment, and sperm-egg mixture treatment after insemination), and treating Hong Kong oyster embryos with six different concentrations of menthol for 15 minutes before artificial insemination, both the treatment method and concentration had varying degrees of influence on the fertilization rate. The egg-only treatment method resulted in the optimal fertilization rate for Hong Kong oyster embryos, with fertilization rates at menthol concentrations of 10-50 mg / L significantly higher than the 0 mg / L control group (P<0.05).

[0065] Table 5. Effects of different concentrations of menthol on hatching rate of Hong Kong oysters under different germ cell treatment methods.

[0066]

[0067] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0068] As shown in Table 5, the experimental results revealed that using four different methods (simultaneous sperm and egg treatment followed by insemination, egg-only treatment, sperm-only treatment, and sperm-egg mixture treatment) and treating Hong Kong oyster embryos with six different concentrations of menthol 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 30 mg / L concentration group exhibiting the highest hatching rate, significantly higher than the 0 mg / L control group (P < 0.05).

[0069] Table 6. Effects of different concentrations of menthol on the malformation rate of Hong Kong oysters under different germ cell treatment methods.

[0070]

[0071] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05).

[0072] As shown in Table 6, the experimental results indicate that using four different methods (simultaneous sperm and egg treatment before fertilization, egg-only treatment, sperm-only treatment, and sperm-egg mixture treatment after insemination), and treating Hong Kong oyster embryos with six different concentrations of menthol 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 20-30 mg / L concentration group showing the lowest malformation rate, significantly lower than the 0 mg / L control group (P<0.05).

[0073] 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 concentrations of menthol; Figure 2 A indicates simultaneous sperm and egg re-fertilization treatment (spermandeggs); Figure 2 B indicates that only eggs are processed. Figure 2 C indicates that only sperm is processed. Figure 2 D indicates post-insemination treatment; such as... Figure 2 As shown in the two-way ANOVA, among the four methods, the method of sperm-egg mixing followed by processing resulted in the lowest rate of malformation in Hong Kong oyster embryos after dissection. Figure 2 C), significantly lower than the other three methods (P<0.05); at the same time, the hatching rate was also at the highest level ( Figure 2 B), but the difference was not significant compared with the other three methods (P>0.05); at the same time, the fertilization rate was also at a relatively high level. Figure 2 A) The difference was significantly higher than that of the method of simultaneous treatment of sperm and egg for re-fertilization (P<0.05), but there was no significant difference compared with the method of treating only eggs and the method of treating only sperm (P>0.05).

[0074] Figure 3The images show photomicrographs of D-type larvae after artificial insemination and hatching using four different methods. Only the experiment on July 16, 2023, from three replicates is shown. As can be seen, among the methods involving sperm-egg mixing and subsequent treatment, the 20-30 mg / L menthol treatment group showed the best hatching results, primarily characterized by more regular D-type larvae shape and a lower proportion of deformed larvae, significantly superior to the control group and other concentration groups using the same method. Combining the analysis of fertilization rate in Table 5, hatching rate in Table 6, and malformation rate in Table 7, under the method of treating Hong Kong oyster embryos with menthol at a concentration of 20-30 mg / L for 15 minutes after sperm-egg mixing and subsequent treatment, followed by dissection, artificial insemination, and hatching, the fertilization rate of the embryos reached a maximum of 70.10%, which was 50.49% higher than the control group; the hatching rate reached a maximum of 77.06%, which was 88.64% higher than the control group; and the malformation rate of the hatched D-type larvae was as low as 11.39%, which was 88.04% lower than the control group.

[0075] In summary, both treatment methods involving menthol on parent and gametes of *Crassostrea gigas* significantly improved embryonic development in Hong Kong oysters. Experiments showed that when treating parent oysters with menthol at a final concentration of 20 mg / L and a seawater salinity of 12‰, the fertilization rate of the treated oysters reached 96.87±3.12%, compared to 46.66±2.31% in the control group; the hatching rate reached 67.75±3.95%, compared to 16.25±4.60% in the control group; and the deformity rate of the hatched D-type larvae was 9.87%. ±0.72%, compared to 50.00±16.67% in the control group; when treating Hong Kong oyster gametes with menthol, the final concentration of menthol was 20-30 mg / L. During treatment, the sperm and eggs of Hong Kong oysters were mixed and soaked in a menthol-infused seawater solution with a salinity of 12‰. The results showed that the fertilization rate of the embryos reached a maximum of 70.10%, which was 50.49% higher than the control group, and the hatching rate reached a maximum of 77.06%, which was 88.64% higher than the control group. The deformity rate of the hatched D-type larvae was as low as 11.39%, which was 88.04% lower than the control group.

[0076] Example 3:

[0077] This embodiment of the study used the same method to react with the Manila clam, whose reproductive mode is similar to that of the oyster, to observe the effect of menthol on the reproduction of the Manila clam. Specifically:

[0078] Treatment 1: The optimal conditions of Example 1 were used, namely, the Manila clam parent clam was soaked in water with a final concentration of 20 mg / L of menthol and a seawater salinity of 12‰. The treatment method is the same as in Example 1. The fertilization rate, hatching rate and deformity rate of the Manila clam were then tested. The conditions of seawater salinity of 12‰ and menthol concentration of 0 mg / L were used as control group 1.

[0079] Treatment 2: The optimal conditions of Example 2 were adopted, namely: the sperm and eggs of Manila clams were mixed and then soaked in a solution of menthol diluted to a final concentration of 30 mg / L with a seawater salinity of 12‰. Other treatment methods are as described in Example 1. Control group 2 was the mixture of sperm and eggs of Manila clams and then soaked in seawater with a salinity of 12‰ for 15 min. Other treatment methods are as described in Example 1.

[0080] The results are shown in Table 7.

[0081] Table 7 Effects of menthol on the reproductive performance of Manila clams

[0082] Group fertilization rate Hatching rate Deformity rate Process 1 <![CDATA[20.56±3.61 b ]]> <![CDATA[16.58±3.58 b ]]> <![CDATA[96.31±4.16 a ]]> Control group 1 <![CDATA[45.23±2.36 a ]]> <![CDATA[40.69±8.14 a ]]> <![CDATA[63.52±2.06 b <!-- 7 -->]]> Process 2 <![CDATA[23.13±6.98 b ]]> <![CDATA[23.67±3.81 b ]]> <![CDATA[93.54±1.97 a ]]> Control group 2 <![CDATA[43.69±5.29 a ]]> <![CDATA[43.65±2.69 a ]]> <![CDATA[53.26±5.13 c ]]>

[0083] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0084] As shown in Table 7, when treated with the same conditions, the fertilization rate and hatching rate of Manila clams in both treatment 1 and treatment 2 were significantly lower than those in the control group, while the malformation rate was significantly higher. This indicates that although different animals have similar reproductive methods, the effects of menthol on their reproductive performance are different due to species differences. In the breeding of Manila clams, menthol did not improve the embryonic development ability of Manila clams.

[0085] In summary, this indicates that menthol has an effect on improving the reproductive performance of Hong Kong oysters, but it may not have the same effect on other animals.

[0086] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Use of menthol for improving embryonic development capacity of Crassostrea hongkongensis, characterized in that, The menthol concentration is 20-30 mg / L.

2. The method of claim 1 for using menthol to enhance the embryonic development capacity of Crassostrea hongkongensis, characterized in that, The method comprises treating the parent Hong Kong oyster with menthol or treating the Hong Kong oyster gamete with menthol; the menthol concentration is 20-30 mg / L; the seawater salinity when the parent Hong Kong oyster is treated is 12‰; the Hong Kong oyster gamete is treated after mixing sperm and egg and then soaked in menthol.

3. The method of claim 2, wherein, The method for treating the parent Hong Kong oyster comprises the following specific steps: S1, collecting and temporarily cultivating the parent Hong Kong oyster: collecting the parent Hong Kong oyster in the breeding peak period and placing it in a cultivation pond for temporary cultivation; S2, preparation before treatment: preparing three cement pools in advance with fresh filtered seawater, adjusting the salinity to 12‰, adding menthol in the first cement pool in advance, and adjusting the concentration to 20 mg / L; S3, menthol treatment: the Hong Kong oyster parents of step S1 are put into the first cement pool of step S2, the seawater temperature is 27-30℃, the salinity is 12‰, the menthol concentration is 20 mg / L, after 24 h of treatment, they are transferred to the second cement pool, the seawater temperature, salinity and menthol concentration in the pool are consistent with those of the first cement pool, after 24 h of continuous treatment in the second cement pool, they are transferred to the third cement pool for another 24 h of treatment, and the Hong Kong oyster parent density is 200 kg / m 3 , and the total treatment time is 72 h; S4, artificial breeding: dissecting all the parent Hong Kong oysters treated in S3, performing artificial insemination and hatching by the traditional method, and the seawater salinity during hatching is 12‰.

4. The method of claim 2, wherein, The method for treating the Hong Kong oyster gamete comprises the following: ① Obtaining the Hong Kong oyster gamete: dissecting the parent Hong Kong oyster temporarily cultivated in the dissection room for 3-15 days and in the breeding period to obtain sperm and egg; ② Menthol treatment: mixing the sperm and egg quantitatively and standing for 10 minutes, then transferring the sperm-egg mixture to 12‰ seawater with 20-30 mg / L menthol for treatment for 15 minutes; ③ Elution: after treatment for 15 minutes in step ②, rapidly eluting the sperm-egg mixture in step ② with a 400-mesh silk screen to wash away the treatment liquid; ④ Hatching: placing the sperm-egg mixture eluted in step ③ in seawater with a salinity of 12‰ for hatching.

5. The method of claim 4, wherein, The method of quantifying the sperm and egg in step ② is diluting the sperm and the egg respectively with seawater with a salinity of 12 ‰ and a water temperature of 30 ℃, 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.

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

1.

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

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