Method for improving reproductive performance of hong kong oyster by l-acetylcarnitine and application thereof

Abstract

The present application relates to the aquaculture technical field, especially relates to a method for improving the reproductive performance of Hongkong oyster by L-acetyl carnitine and application, the present application adopts two ways of L-acetyl carnitine treatment Hongkong oyster parent and Hongkong oyster gamete to treat Hongkong oyster, and the reproductive performance of Hongkong oyster is significantly improved in the two ways, and the hatching rate is significantly improved;Through experiment, when the parent is treated by L-acetyl carnitine with a final concentration of 150mg / L under the condition that the salinity of seawater is 12‰, the hatching rate of oyster is improved from 16.25% of the control group to 71.88%, which is increased by 3.42 times;When the sperm and egg of oyster are mixed and soaked for 15min by L-acetyl carnitine with a final concentration of 100mg / L under the condition that the salinity of seawater is 12‰, the hatching rate of oyster is improved from 49.78% of the control group to 88.95%, which is increased by 78.69%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of aquaculture technology, in particular to a method for improving the reproductive performance of Crassostrea hongkongensis by L-acetylcarnitine and application thereof. BACKGROUND

[0002] Crassostrea hongkongensis, commonly known as large oysters, is mainly distributed in the brackish water area of river estuary and is one of the most important economic shellfishes in the coastal areas of southern China. Under the influence of human development and climate change, the reproductive performance of the parent oysters in the natural sea area is increasingly deteriorating, mainly manifested in the problems of natural discharge barrier of male and female gametes, low fertilization rate, low hatching rate, high deformity rate, and dramatic decrease in the number of D-type larvae, which further leads to the degradation of germplasm resources. It can be said that the increasingly deteriorating reproductive performance has seriously threatened the survival of wild resources of Crassostrea hongkongensis and the development of the oyster farming industry.

[0003] In view of the problem of natural discharge barrier of male and female gametes of Crassostrea hongkongensis, in the practice of artificial seed production, the method of dissecting and collecting sperm and eggs for artificial insemination and hatching is usually adopted, which solves the problem of discharge barrier, but the maturity of the sperm and eggs obtained by dissection is inconsistent, which still leads to low fertilization rate, low hatching rate, high deformity rate, and low number of D-type larvae, and low hatching efficiency.

[0004] In northern China, the method of promoting the maturation of the gonad of long oysters by increasing the temperature and nutritional enrichment has been used to improve the maturity and has achieved good hatching results. However, the Hong Kong oysters grown in Guangdong and Guangxi have been living in high water temperature for a long time, that is, the biological zero is higher, and the range of temperature increase is limited, so the effect of temperature increase on maturation is poor. Nutritional enrichment also needs to be carried out in low water temperature to achieve better results, which is not suitable for the high water temperature in South China. The main reasons are as follows: first, the water temperature in Guangdong and Guangxi is high, and the Hong Kong oysters have fast metabolism, so it is not easy to accumulate nutrients and convert them into reproductive performance. Second, if some high-nutrient 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 nutritional enrichment, but also causes a large number of deaths.

[0005] L-acetylcarnitine is an important carnitine derivative developed by Sigma-Tau Company in Italy. It was launched in 1986 and is used for the treatment of central nervous system diseases, metabolic diseases, dementia and brain aging, etc. L-acetylcarnitine can also be converted into other carnitine compounds in the body, thereby playing an important role. In addition, L-acetylcarnitine has certain antioxidant function and anti-aging function, and has the possibility of protecting cells such as ova and sperm, but there is no report on the use of L-acetylcarnitine to improve the reproductive performance of oysters in the prior art. However, based on its antioxidant capacity, it is possible to improve the reproductive performance of oysters and improve the survival rate of ova, and can be used to process a promoter for promoting the reproduction of Hong Kong oysters. SUMMARY

[0006] In view of the above, in view of the deficiencies of the prior art, the present application provides a new method for improving the reproductive performance of Hong Kong oysters in a short period of time using L-acetylcarnitine, which solves the problems of low efficiency of dissection and collection of sperm and ova, and the unsuitability of traditional warming and nutrient enrichment methods for artificial breeding of Hong Kong oysters in the south. Adapted to local conditions, the reproductive performance of Hong Kong oyster parents and gametes is effectively improved, 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 L-acetylcarnitine in improving the reproductive performance of Hong Kong oysters, wherein the concentration of L-acetylcarnitine is 100mg / L-150mg / L.

[0009] The application also includes a method for improving the reproductive performance of Hong Kong oysters using L-acetylcarnitine according to claim 1, which comprises treating Hong Kong oyster parents with L-acetylcarnitine and / or treating Hong Kong oyster gametes with L-acetylcarnitine; the concentration of L-acetylcarnitine is 100mg / L-150mg / 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 ova for soaking.

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

[0011] S1, collecting and temporarily raising Hong Kong oyster parents: collecting Hong Kong oyster parents in the breeding period and placing them in a breeding cage and a breeding pond for temporary raising;

[0012] S2, preparation before treatment: three cement pools with fresh filtered seawater are prepared in advance, and the salinity of seawater in the three cement pools is adjusted to 12‰. L-acetylcarnitine is added to the first cement pool in advance, and the concentration is adjusted to 150mg / L.

[0013] S3, L-acetylcarnitine treatment: the parent oysters of Hong Kong oyster in step S1 are put into the first cement pool in step S2 for treatment for 24 hours; L-acetylcarnitine is added into the second cement pool to a concentration of 150 mg / L, and the parent oysters treated for 24 hours are put into the second cement pool for treatment for 48 hours; L-acetylcarnitine is added into the third cement pool to a concentration of 150 mg / L, and the parent oysters treated for 24 hours are put into the third cement pool for treatment for 24 hours, and the total treatment time is 72 hours; the parent oysters of Hong Kong oyster are put into the cement pool at a density of 150-250 kg / m 3 ;

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

[0015] Further, the seawater temperature in step S2 is 27-30℃.

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

[0017] ① obtaining the gametes of Hong Kong oyster: after the parent oysters of Hong Kong oyster which are temporarily cultured in the room for 3-15 days and are in the breeding period, the male and female are distinguished, and the sperm and egg are obtained by dissection, respectively;

[0018] ② L-acetylcarnitine treatment: after the sperm and egg are quantitatively mixed and left for 10 minutes, the mixture is transferred to a container containing L-acetylcarnitine treatment solution with a salinity of 12 ‰, and the final concentration of L-acetylcarnitine in the container is 100 mg / L, and the treatment time is 15 minutes;

[0019] ③ elution: after the treatment for 15 minutes in step ②, the mixture of sperm and egg in step ② is eluted quickly with a 400-mesh silk screen to wash off the treatment solution;

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

[0021] Further, the method for quantitatively mixing the sperm and egg in step ② is to dilute the sperm and egg with seawater with a salinity of 12 ‰ and a water temperature of 30℃, and 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 10 L; the density of the sperm mother liquor is 10 2 -10 3 pieces / microliter, and the total volume is 10 L.

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

[0023] Further, the incubation temperature of step 4 is 27-30℃.

[0024] In the present application, the density of S2 step has the following corresponding relationship with seawater temperature:

[0025] Water temperature 27℃ 28℃ 29℃ 30℃ Density 250 kg / m 3 ]] 225 kg / m 3 ]] 200 kg / m 3 ]] 150 kg / m 3 ]]

[0026] The present application has the following beneficial effects:

[0027] 1. The present application significantly improves the reproductive performance of Hong Kong oysters by using L-acetylcarnitine of appropriate concentration, treating the parents of Hong Kong oysters under appropriate seawater salinity, and treating the gametes of Hong Kong oysters under appropriate methods, which greatly improves the hatching efficiency in artificial breeding production of Hong Kong oysters. Experimental data show that when the concentration of L-acetylcarnitine is 150 mg / L, the parents of Hong Kong oysters are treated under 12‰ seawater salinity, and then artificial insemination and hatching are performed, the reproductive performance reaches the optimum, the fertilization rate reaches 90.92%, which is 94.86% higher than that of the control group, the hatching rate reaches 71.88%, which is 3.42 times higher than that of the control group, and the deformity rate of D-type larvae is not different from that of the control group. In the method of treating the gametes of Hong Kong oysters, after the sperm and eggs are mixed, the method of treating with 100 mg / L concentration of L-acetylcarnitine for 15 minutes is used, the fertilization rate of Hong Kong oyster embryos reaches 82.40%, which is 81.86% higher than that of the control group, the hatching rate reaches 88.95%, which is 78.69% higher than that of the control group, and the deformity rate of D-type larvae is 6.17%, which is 89.09% lower than that of the control group. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 1 is a double-factor variance analysis diagram of L-acetylcarnitine treatment of Hong Kong oyster parents under different seawater salinity and different concentration gradients; Figure 1 A is the fertilization rate result diagram; Figure 1 B is the hatching rate result diagram; Figure 1 C is the deformity rate result diagram.

[0029] Figure 2 Figure 2 is a double-factor variance analysis diagram of L-acetylcarnitine treatment of Hong Kong oyster reproductive cells under four different treatment methods and different concentration gradients; Figure 1 A is the fertilization rate result diagram; Figure 1 B is the hatching rate result diagram; Figure 1 C is the deformity rate result diagram.

[0030] Figure 3 Figure 3 is a microscopic photograph of D-type larvae after four methods of treatment and artificial insemination and hatching. DETAILED DESCRIPTION

[0031] All features disclosed in this specification, and / or all methods or processes disclosed in this specification may be combined in any combination, provided such values, combinations thereof, and / or methods or processes are not mutually inconsistent.

[0032] Any of the features disclosed in this specification, unless explicitly stated otherwise, are examples of a generic series of equivalent or similar features.

[0033] Example 1

[0034] This example studies the effects of different concentrations of L-acetyl carnitine on the reproductive performance of Hong Kong oyster parents under different salinities through a two-factor experiment, including the following steps:

[0035] S1, collect and temporarily raise Hong Kong oyster parents: In June 2022, 300 kg of 3-year-old Hong Kong oyster parents with a size of 8.57±1.32 cm were collected in a hatchery, after cleaning the surface attachments and sludge, they were respectively placed in 12 culture cages in a 20 m cubed water aquaculture pond for temporary raising for 3 days. The salinity of the seawater during temporary raising was 15%, the water temperature was 30℃, and sufficient chlorella was fed and the air was ensured.

[0036] S2, preparation before treatment: considering the economy of drug use, high-density and short-term treatment method is used for treatment, 36 100L treatment containers with fresh filtered seawater are prepared in advance, the water body is 80L, divided into 2 groups: high salinity group and low salinity group, 18 treatment containers in each group, the salinity of seawater in high salinity group is adjusted to 24‰, the salinity of seawater in low salinity group is adjusted to 12‰, the water temperature is 30℃, then L-acetyl carnitine is added in the first 6 treatment containers of each group, the concentration is adjusted to 0mg / L, 50mg / L, 100mg / L, 150mg / L, 200mg / L, and 250mg / L respectively; the seawater temperature in this step is maintained at 27-30℃.

[0037] S3, L-acetyl carnitine treatment: the 12 culture cages containing Hong Kong oyster parents were respectively placed in the 6 concentration gradient treatment containers of the high salinity group and the low salinity group for the first treatment, and the Hong Kong oyster parent density was 150kg / m 3, after 24h treatment, the 2nd treatment was performed; the 2nd treatment was performed according to the S2 method, and L-acetylcarnitine was added into another 12 treatment containers in each group, and the final concentration of L-acetylcarnitine corresponded to the 1st treatment (adjusted to 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, respectively); after 24h treatment, the 3rd treatment was performed; the 3rd treatment was performed according to the S2 method, and L-acetylcarnitine was added into another 12 treatment containers in each group, and the final concentration of L-acetylcarnitine corresponded to the 1st treatment (adjusted to 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, respectively); after 24h treatment, the treatment process was ended, the total treatment time was 72h, and there was no water change during the treatment, sufficient Chlorella was fed, and aeration was maintained.

[0038] S4, artificial breeding: after the high-salt group and the low-salt group and the different concentration gradient groups of Hong Kong oysters were dissected and distinguished as male and female, the traditional method was used for in-group artificial insemination and hatching, the seawater salinity during hatching was 12‰, the seawater temperature was 27-30℃, and the fertilization rate, hatching rate and deformity rate of the high-salt group and the low-salt group under different concentration gradients were measured, and two-factor variance analysis was performed. Among them, the calculation method of fertilization rate, hatching rate and deformity rate is:

[0039] ① Fertilization rate (%) = number of fertilized eggs / total number of eggs x 100;

[0040] ② Hatching rate (%) = number of hatched larvae / number of fertilized eggs x 100;

[0041] ③ Deformity rate (%) = number of deformed larvae / number of hatched larvae x 100.

[0042] The density of the S2 step of the example has the following corresponding relationship with seawater temperature:

[0043] Table 1 Corresponding relationship between seawater temperature and density

[0044] Water temperature 27℃ 28℃ 29℃ 30℃ Density 250 kg / m 3 ]] 225 kg / m 3 ]] 200 kg / m 3 ]]> 150 kg / m 3 ]]

[0045] The experiment was repeated 3 times. The statistical results of the fertilization rate, hatching rate and deformity rate obtained are shown in Tables 2-4.

[0046] Table 2 Fertilization rate of Hong Kong oysters after L-acetylcarnitine treatment under different salinities

[0047]

[0048] Note: The same row different lowercase letters indicate the same salinity group, different concentration groups between the fertilization rate difference was significant (P<0.05), * indicates the highest fertilization rate of 12‰ salinity group and the same concentration gradient treatment of 24‰ salinity group difference was significant (P<0.05).

[0049] From the experimental results of Table 2: in different salinity (12‰ and 24‰), 6 concentration gradient L-acetyl carnitine treatment of Hong Kong oyster parents 72 hours, then artificial insemination, seawater salinity and treatment concentration two factors on the fertilization rate produced varying degrees of influence. In 24‰ seawater salinity, 50mg-150mg / L treatment group had the highest fertilization rate; in 12‰ seawater salinity, 100mg / L treatment group had the highest fertilization rate, and was significantly higher than 0mg / L control group (P<0.05).

[0050] Table 3 Hong Kong oyster in different salinity after L-acetyl carnitine treatment of hatching rate

[0051]

[0052] Note: The same row different lowercase letters indicate the same salinity group, different concentration gradient treatment after the hatching rate difference was significant (P<0.05), * indicates the lowest hatching rate of 12‰ salinity group and the same concentration gradient treatment of 24‰ salinity group difference was significant (P<0.05).

[0053] From the experimental results of Table 3: in different salinity (12‰ and 24‰), 6 concentration gradient L-acetyl carnitine treatment of Hong Kong oyster parents 72 hours, then artificial insemination hatching, seawater salinity and treatment concentration two factors on the hatching rate produced varying degrees of influence. In 12‰ seawater salinity, the hatching rate of each treatment group was significantly higher than 24‰ salinity (P<0.05), among which the 150mg / L concentration treatment group had the highest hatching rate, and was significantly higher than 0mg / L control group (P<0.05).

[0054] Table 4 Hong Kong oyster in different salinity after L-acetyl carnitine treatment of deformity rate

[0055]

[0056] Note: The same row different lowercase letters indicate the same salinity group, different concentration gradient treatment after the deformity rate difference was significant (P<0.05), * indicates the lowest deformity rate of 12‰ salinity group and the same concentration gradient treatment of 24‰ salinity group difference was significant (P<0.05).

[0057] As shown in Table 4, under different salinities (12‰ and 24‰), after treating Hong Kong oyster parents with L-acetylcarnitine 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‰, there was no significant difference in deformity rate among the 0 mg-200 mg / L treatment groups (P>0.05), but all were significantly lower than the 250 mg / L treatment group (P<0.05).

[0058] 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 shown in the figure, under a seawater salinity of 12‰, the hatching rate of Hong Kong oyster parents treated with different concentration gradients of L-acetylcarnitine was ( Figure 1 B) Overall, it was superior to the 24‰ seawater salinity group. At a seawater salinity of 12‰, the fertilization rate was highest in the 100 mg / L L-acetylcarnitine treatment group. Figure 1 A), significantly higher than the 0 mg / L control group and other concentration groups (P<0.05); the 150 mg / L L-acetylcarnitine 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); there was no significant difference in the malformation rate among the 0 mg-200 mg / L concentration treatment groups (P>0.05). Figure 1 C). Further analysis of the fertilization rate in Table 2, the hatching rate in Table 3, and the malformation rate in Table 4 indicates that: Treatment of Hong Kong oyster parent stock with L-acetylcarnitine at a concentration of 150 mg / L for 72 hours at a seawater salinity of 12‰, followed by dissection and artificial insemination, resulted in optimal reproductive performance. The fertilization rate reached 90.92±1.12%, a 94.86% increase compared to the control group (46.66±2.31%). The hatching rate reached 71.88±3.30%, a 3.42-fold increase compared to the control group (16.25±4.60%). The malformation rate of the hatched D-type larvae was not different from that of the control group.

[0059] Example 2:

[0060] This invention provides a method for improving the reproductive performance of Hong Kong oysters by treating gametes with L-acetylcarnitine, comprising the following steps:

[0061] ① Obtain gametes of Hong Kong oyster: In July 2022, 20 parent Hong Kong oysters were collected from the temporary breeding room of Guangxi Maowei Sea, the indoor seawater salinity was 12%, the water temperature was 30℃, and sufficient feed was fed. After 15 days of temporary breeding, the gametes of Hong Kong oysters were obtained by dissecting the 20 parent Hong Kong oysters after distinguishing male and female, and the sperm and eggs were quantified, wherein the quantification method was: the sperm and eggs were diluted with seawater with a salinity of 12‰ and a water temperature of 30℃, and finally the sperm mother liquor and egg mother liquor were obtained, wherein the density of the egg mother liquor was 50 pieces / ml, and the total volume was 10L; the density of the sperm mother liquor was 10 2 -10 3 pieces per microliter, and the total volume was 10L.

[0062] ② L-acetyl carnitine treatment: the gametes of Hong Kong oysters were treated with 4 different treatment methods and 6 different concentration gradients of L-acetyl carnitine, and the treatment time was 15 minutes;

[0063] ③ Elution: after 15 minutes of treatment, the embryos were quickly eluted (wash the treatment solution) with a 400-mesh silk screen;

[0064] ④ Incubation: the newly eluted treatment solution was placed in a fresh seawater container with a salinity of 12‰ and a water temperature of 27-30℃ for incubation, the incubation process was trace aeration, and the fertilization rate, hatching rate and malformation rate were measured, and two-factor variance analysis was performed.

[0065] In step 2, the four different treatment methods are as follows: (1) Simultaneous treatment of sperm and eggs (sperm and eggs), that is, after treating sperm and eggs with different concentration gradients of L-acetylcarnitine for 15 minutes, artificial insemination is performed. The specific operation is as follows: 12 plastic beakers are prepared, and 6 different concentration gradients of L-acetylcarnitine 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 treating the eggs and sperm 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 treat the eggs (eggs only), that is, only treat the eggs with different concentration gradients of L-acetylcarnitine for 15 minutes, then add untreated sperm for artificial insemination. The specific operation is as follows: 6 plastic beakers are prepared, and 6 different concentration gradients of L-acetylcarnitine 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 treat the sperm (sperm only), that is, only treat the sperm with different concentration gradients of L-acetylcarnitine for 15 minutes, then add untreated eggs 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; 6 different concentration gradients of L-acetylcarnitine 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 the untreated eggs, and incubation is performed; (4) After insemination, that is, after treating the fertilized eggs with different concentration gradients of L-acetylcarnitine for 15 minutes, incubation is performed. The specific operation is as follows: 12 plastic beakers are prepared, and 6 different concentration gradients of L-acetylcarnitine 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 were added to the corresponding treatment solution, and after 15 minutes of treatment in the treatment solution, the eggs were washed (same method as above) and placed in a beaker of fresh seawater for incubation.

[0066] In step 2, the 6 different concentration gradients of L-acetylcarnitine treatment are: 0 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, and 250 mg / L.

[0067] This experiment was conducted in three different periods (July 16, 2023, July 18, 2023, and July 21, 2023) with three replicates. The final statistical results of fertilization rate, hatching rate and abnormality rate are shown in Tables 5-7.

[0068] Table 5 Effect of different concentrations of L-acetylcarnitine on the fertilization rate of Hong Kong oyster gametes under different treatment methods

[0069]

[0070] Note: The same row with different lowercase letters indicates that the fertilization rate after treatment with different concentration gradients in the same method group is significantly different (P<0.05), and * indicates that the highest fertilization rate after treatment of the sperm-egg mixture is significantly different from that of other method groups with the same concentration (P<0.05).

[0071] From the experimental results in Table 5, it can be seen that using four different methods (simultaneous treatment of sperm and egg, treatment of egg only, treatment of sperm only, and treatment of sperm-egg mixture), and six different concentrations of L-acetylcarnitine to treat Hong Kong oyster dissected embryos for 15 minutes, and then artificially inseminated, the treatment method and treatment concentration both had different effects on the fertilization rate. In the methods of treating sperm only and treating sperm-egg mixture, the fertilization rate of Hong Kong oyster embryos reached the optimal level. In the method of treating sperm only, the fertilization rate of L-acetylcarnitine at concentrations of 50-200 mg / L was better, significantly higher than that of the control group (P<0.05). In the method of treating sperm-egg mixture, the treatment effect of L-acetylcarnitine at concentrations of 50-100 mg / L was better, significantly higher than that of the control group (P<0.05).

[0072] Table 6 Effect of different concentrations of L-acetylcarnitine on the hatching rate of Hong Kong oyster gametes under different treatment methods

[0073]

[0074] Note: The same row with different lowercase letters indicates that the hatching rate after treatment with different concentration gradients in the same method group is significantly different (P<0.05), and * indicates that the highest hatching rate after treatment of the sperm-egg mixture is significantly different from that of other method groups with the same concentration (P<0.05).

[0075] 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 L-acetylcarnitine 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 100-150 mg / L concentration group showing the highest hatching rate, significantly higher than the 0 mg control group (P<0.05).

[0076] Table 7. Effects of different concentrations of L-acetylcarnitine on gamete malformation rate in Hong Kong oysters under different germ cell treatment methods.

[0077]

[0078] Note: Different lowercase letters in the same row indicate that the difference in deformity rate after different concentration gradient treatments within the same method group is significant (P < 0.05). * indicates that the lowest deformity rate of the sperm-egg mixed treatment group is significantly different from that of other method groups treated with the same concentration (P < 0.05).

[0079] As shown in Table 7, treating Hong Kong oyster embryos with six different concentrations of L-acetylcarnitine for 15 minutes using four different methods before artificial insemination and hatching resulted in varying degrees of influence on the malformation rate. The malformation rate of Hong Kong oyster embryos was lowest when treated after sperm-egg mixing, with the 100 mg / L concentration group showing the lowest rate, significantly lower than the 0 mg control group (P<0.05).

[0080] A two-way ANOVA was performed on the above experimental results, and the results are as follows: Figure 2 As shown: Different colored bars in the figure represent different L-acetylcarnitine concentration groups; the four clusters on the horizontal axis represent simultaneous sperm and egg treatment before fertilization (labeled "spermand eggs"), egg treatment only (labeled "eggs only"), sperm treatment only (labeled "sperm only"), and sperm and egg mixing treatment (labeled "after insemination"); the vertical axis represents the fertilization rate (%). Figure 2 A) Hatching rate (%) Figure 2 B) and deformity rate (%) Figure 2 C). For example Figure 2 As shown, after comprehensive analysis using two-way ANOVA, among the four methods, the method of treating oysters after sperm-egg mixing resulted in the highest hatching rate of dissected gametes from Hong Kong oysters. Figure 2B), significantly higher than the method of simultaneous treatment of sperm and egg (P<0.05), but not significantly different from the method of only treating eggs and the method of only treating sperm (P>0.05); at the same time, the fertilization rate was also at a higher level Figure 2 A), significantly higher than the method of simultaneous treatment of sperm and egg (P<0.05), but not significantly different from the method of only treating eggs and the method of only treating sperm (P>0.05); at the same time, the deformity rate was at a lower level Figure 2 C), but not significantly different from the other three methods (P>0.05).

[0081] Figure 3 Micrographs of D-type larvae after artificial insemination and incubation of four methods, micrographs of D-type larvae of each method group and concentration group (only 2023-07-16 experiments in three repeated experiments are shown), it can be seen from the figure that in the method of treating after mixing sperm and egg, the treatment group of L-acetylcarnitine with a concentration of 100-150 mg / L has the best hatching effect, mainly manifested as D-type larvae (marked as D-larvae in the figure) with more regular shape and lower proportion of malformation larvae (marked as malformation larvae in the figure), which is significantly better than the control group (marked as Control in the figure) 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, it is considered that under the method of treating after mixing sperm and egg, the reproductive performance is optimal after treating Hong Kong oyster embryos with L-acetylcarnitine with a concentration of 100 mg / L for 15 minutes and then dissecting and artificially inseminating and incubating, in which the fertilization rate of Hong Kong oyster embryos reaches 82.40%, which is increased by 81.86% compared with the control group, the hatching rate reaches 88.95%, which is increased by 78.69% compared with the control group, and the deformity rate of D-type larvae after hatching is 6.17%, which is decreased by 89.09% compared with the control group.

[0082] In summary, both of the two ways of treating Hong Kong oysters by using L-acetyl carnitine can significantly improve the reproductive performance of Hong Kong oysters. According to the experiment, when the parent Hong Kong oysters are treated by using L-acetyl carnitine, the final concentration of L-acetyl carnitine is 150 mg / L, and the salinity of seawater is 12‰. It is verified that the fertilization rate of the treated Hong Kong oysters reaches 90.92%, which is increased by 94.86% compared with the control group. The hatching rate reaches 71.88%, which is increased by 3.42 times compared with the control group. There is no difference in the deformity rate of D-type larvae between the treated Hong Kong oysters and the control group. When the gametes of Hong Kong oysters are treated by using L-acetyl carnitine, the final concentration of L-acetyl carnitine is 100 mg / L. The sperm and eggs of Hong Kong oysters are mixed and then soaked in the L-acetyl carnitine seawater solution. The salinity of seawater is 12‰. It is verified that the fertilization rate of the treated Hong Kong oyster embryos reaches 82.40%, which is increased by 81.86% compared with the control group. The hatching rate reaches 88.95%, which is increased by 78.69% compared with the control group. The deformity rate of D-type larvae is 6.17%, which is decreased by 89.09% compared with the control group.

[0083] Example 3

[0084] In this example, the same method is used to treat Ruditapes philippinarum, which has similar reproductive methods and overlapping natural distribution with Hong Kong oysters. The effect of L-acetyl carnitine on the reproduction of Ruditapes philippinarum is observed. Specifically,

[0085] Treatment 1: The optimal conditions of Example 1 are used, i.e., the parent Ruditapes philippinarum is soaked under the condition that the final concentration of L-acetyl carnitine is 150 mg / L and the salinity of seawater is 12‰. The treatment method is referred to Example 1. The fertilization rate, hatching rate and deformity rate of Ruditapes philippinarum are detected. The condition that the salinity of seawater is 12‰ and the concentration of L-acetyl carnitine is 0 mg / L is used as control group 1.

[0086] Treatment 2: The optimal conditions of Example 2 are used, i.e., the sperm and eggs of Ruditapes philippinarum are mixed and then soaked in the solution with the salinity of seawater being 12‰ and the final concentration of L-acetyl carnitine being 100 mg / L. The other treatment methods are referred to Example 1. The sperm and eggs of Ruditapes philippinarum are mixed and then soaked in seawater with the salinity of 12‰ for 15 min as control group 2. The other treatment methods are referred to Example 1.

[0087] The results are shown in Table 8.

[0088] Table 8 Effect of L-acetyl carnitine on the reproductive performance of Ruditapes philippinarum

[0089] Group Fertilization rate Hatching rate Malformation rate Treatment 1 36.58 ± 2.98 b ]] 32.98 ± 2.36 b ]] 82.69 ± 8.25 a ]] Control group 1 45.23 ± 2.36 a ]] 40.69 ± 8.14 a ]] 63.52 ± 2.06 b ]] Treatment 2 32.98 ± 1.58 b ]] 30.25 ± 9.68 b ]] 83.68 ± 0.69 a ]] Control group 2 43.69 ± 5.29 a ]] 43.65 ± 2.69 a ]] 53.26 ± 5.13 c ]]

[0090] Note: Different lowercase letters in the same column represent significant differences (P<0.05).

[0091] From Table 8, it can be seen that, after the same treatment of Manila clams, the fertilization rate and hatching rate of Manila clams in treatment 1 and treatment 2 are significantly lower than those of the control group, and the deformity rate is significantly increased, which shows that, for different animals, although the reproductive modes are similar, due to the difference in species, the influence of L-acetyl carnitine on the reproductive performance is also different. In the breeding of Manila clams, L-acetyl carnitine cannot improve the reproductive performance of parents or gametes.

[0092] In summary, it is shown that L-acetyl carnitine has an improvement effect on the reproductive performance of Hong Kong oysters, but may not have the same effect on other animals.

[0093] The above-described examples only express several embodiments of the present application, and the description is relatively specific and detailed, but it cannot be understood as a limitation on 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, several modifications and improvements can be made, which belong to the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the appended claims.

Claims

1. Use of L-acetyl carnitine for improving the reproductive performance of Crassostrea hongkongensis, characterized in that, The L-acetylcarnitine concentration is 100-150 mg / L.

2. The method for enhancing the reproductive performance of Hong Kong oysters using L-acetylcarnitine as described in claim 1, characterized in that, The method comprises treating the parent oysters of Crassostrea hongkongensis with L-acetylcarnitine and / or treating the gametes of the Crassostrea hongkongensis with L-acetylcarnitine; the L-acetylcarnitine concentration is 100-150 mg / L; the seawater salinity for treating the parent oysters of the Crassostrea hongkongensis is 12‰; and the gametes of the Crassostrea hongkongensis are treated by mixing sperm and eggs for soaking.

3. The method of claim 2, wherein, The method for treating the parent oysters of the Crassostrea hongkongensis comprises the following specific steps: S1, collecting and temporarily cultivating the parent oysters of the Crassostrea hongkongensis: collecting the parent oysters of the Crassostrea hongkongensis in the breeding peak period, putting them into a cultivation cage and placing them in a cultivation pond for temporary cultivation; S2, preparation before treatment: preparing three cement pools with fresh filtered seawater in advance, adjusting the seawater salinity of the three cement pools to 12‰, adding L-acetylcarnitine in the first cement pool in advance, and adjusting the concentration to 150 mg / L; S3, L-acetylcarnitine treatment: the parent oysters of Hong Kong oyster in step S1 were put into the first cement pool for treatment for 24 h; L-acetylcarnitine was added into the second cement pool to a concentration of 150 mg / L, and the parent oysters treated for 24 h were put into the second cement pool for treatment for 48 h; L-acetylcarnitine was added into the third cement pool to a concentration of 150 mg / L, and the parent oysters treated for 24 h were put into the third cement pool for treatment for 24 h, and the total treatment time was 72 h; the parent oysters of Hong Kong oyster were put into the cement pool at a density of 150-250 kg / m 3 ; S4, artificial breeding: after the treated parent oysters of the Crassostrea hongkongensis are all dissected and the male and female are distinguished, the traditional method is adopted for artificial insemination and hatching, and the seawater salinity during hatching is 12‰.

4. The method of claim 3, wherein, The seawater temperature of the S2 step is 27-30℃.

5. The method of claim 2, wherein, The method for treating the gametes of the Crassostrea hongkongensis comprises the following specific steps: ① obtaining the gametes of the Crassostrea hongkongensis: after the parent oysters of the Crassostrea hongkongensis that have been temporarily cultivated indoors for 3-15 days and are in the breeding period are dissected after the male and female are distinguished, sperm and eggs are obtained; ② L-acetylcarnitine treatment: after the sperm and eggs are mixed and placed for 10 minutes, the sperm and egg mixture is transferred to a container containing L-acetylcarnitine treatment liquid with a salinity of 12‰, the final concentration of L-acetylcarnitine in the container is 100 mg / L, and the treatment time is 15 minutes; ③ elution: after 15 minutes of treatment in step ②, the sperm and egg mixture in step ② is eluted with a 400-mesh silk screen to wash away the treatment liquid; ④ hatching: the sperm and egg mixture 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 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.

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

1.

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

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