Method for gender reversal in crustaceans

Abstract

The present application relates to the field of animal breeding, and particularly to a method for gender reversal of crustacean animals. The present application provides a method for gender reversal of crustacean animals, taking crustacean animals after metamorphosis, feeding with steroid hormones, and breeding. The experiment proves that sex hormones of vertebrates play a role in gender differentiation of crustacean animals, and when the concentration of MT added in feed is 2000 mg / kg, PL2 and PL25 male juvenile prawns can be induced; when the concentration of E2 added in feed is 2000 mg / kg, PL25 juvenile prawns can be induced to be female, and male juvenile prawns of PL30 can be reversed to female prawns. The experiment determines the best gender reversal period and hormone concentration in different development stages of juvenile prawns, and observes the egg-carrying "pseudo-female prawns", which lays a foundation for determining the chromosome pairing of juvenile prawns and monosex breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of animal breeding, in particular to a method for gender reversal of crustaceans. BACKGROUND

[0002] The scientific name of the freshwater shrimp is Macrobrachium nipponense, which is the highest yield of freshwater shrimp in China. The premature maturation of the gonad of the small shrimp in the breeding pond leads to a series of problems such as small individual, excessive pond breeding density, and multi-generation in the same room, which restricts the sustainable and healthy development of the freshwater shrimp industry. Therefore, it is urgent to use gender control technology to carry out monosex rearing and cultivate all-female / all-male populations to improve the yield of breeding.

[0003] Steroid hormones mainly exist in testes, ovaries and hepatopancreas, and play an important role in regulating the development of gonad, sex determination and growth of aquatic species through interaction with endocrine factors. Adding steroid hormones in feed is one of the means of gender reversal, which has been achieved in many aquatic animals. For example, feeding 500mg / kg 17α-methyltestosterone to female Siniperca chuatsi can completely reverse the gender of 14.7% of the fish after 40 days. Similarly, feeding 30mg / kg and 60mg / kg 17β-estradiol to brown trout can make the proportion of females in the experimental group reach 84% and 86% after 456 days.

[0004] Under certain conditions, the phenomenon of mutual transformation of male and female individuals of animals is called sex reversal. Fish sex reversal is relatively common, such as the rice field eel. There are cases of using steroid hormones such as androgen: 17α-methyltestosterone (MT) and estrogen: 17β-estradiol (E2) to induce sex reversal in other fish, but there is no successful example of using vertebrate sex hormones in crustaceans. SUMMARY

[0005] Therefore, the present application provides a method for gender reversal of crustaceans. The experiment proves that vertebrate sex hormones play a role in the gender differentiation of crustaceans. When the concentration of MT added in the feed is 2000mg / kg, it can induce the masculinization of PL2 and PL25 freshwater shrimps; when the concentration of E2 added in the feed is 2000mg / kg, it can induce the feminization of PL25 freshwater shrimps and reverse the male PL30 freshwater shrimps to female shrimps. The experiment determines the best period of gender reversal and hormone concentration for different development stages of freshwater shrimps, and observes the "pseudo-female shrimps" that carry eggs, which lays a foundation for determining the chromosome pairing of freshwater shrimps and monosex rearing.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a method for gender reversal of crustaceans, taking crustaceans after metamorphosis, feeding steroid hormones, and breeding.

[0008] In some embodiments of the present application, in the above method, the feeding time is the 2nd day, the 25th day and / or the 30th day after metamorphosis.

[0009] In some embodiments of the present application, in the above method, the steroid hormone comprises: androgen and / or estrogen.

[0010] In some embodiments of the present application, in the above method, the androgen comprises: 17α-methyltestosterone; the estrogen comprises: 17β-estradiol.

[0011] In some embodiments of the present application, in the above method, the concentration of the steroid hormone is: 500-2000 mg / kg.

[0012] In some embodiments of the present application, in the above method, when the feeding time is the 30th day after metamorphosis, the crustacean is divided into female and male; the female is fed with the androgen, and the male is fed with the estrogen.

[0013] In some embodiments of the present application, in the above method, when the feeding time is the 2nd day or the 25th day after metamorphosis, the breeding time is 40 days.

[0014] When the feeding time is the 30th day after metamorphosis, the breeding time is 50 days.

[0015] In some embodiments of the present application, in the above method, the feeding weight is 2% of the body weight of the crustacean.

[0016] In some embodiments of the present application, in the above method, the breeding temperature is 27-29℃, and the oxygen concentration is ≥6 mg / L.

[0017] In some embodiments of the present application, in the above method, the crustacean comprises: shrimp.

[0018] In some embodiments of the present application, in the above method, the crustacean comprises: Chinese shrimp.

[0019] The present application provides a method for gender reversal of crustacean, taking crustacean after metamorphosis, feeding with steroid hormone, and breeding.

[0020] The experiment proves that the vertebrate sex hormone plays a role in the gender differentiation of crustaceans. When the concentration of MT added in the feed is 2000 mg / kg, the PL2 and PL25 juvenile male can be induced. When the concentration of E2 added in the feed is 2000 mg / kg, the PL25 juvenile female can be induced, and the male juvenile of PL30 can be reversed to female. The experiment determines the best gender reversal period and hormone concentration of juvenile at different development stages, and the egg-carrying "pseudo-female" is observed, which lays a foundation for determining the chromosome pairing of juvenile and monosex culture. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description.

[0022] Figure 1 The figure shows the experimental flow chart of using different concentrations of MT and E2 to feed PL2 and PL25 juvenile. Wherein: Post-larvae (PL) refers to metamorphic development; PL2 and PL25 are the second day and the 25th day after metamorphosis. The juvenile starts to feed the feed at the second day after metamorphosis, and the gonadal development is complete but the secondary sexual characteristics are not obvious at the 25th day after metamorphosis, so it is difficult to distinguish male and female; Treatment refers to the concentration of hormone used in the experimental group; Sex ratio refers to the gender ratio;

[0023] Figure 2 The figure shows the gender ratio of PL2 juvenile fed with 1000 mg / kg MT and E2 for 40 days. Wherein: 17α-Methyltestosterone (MT) is shown on the left; 17β-Estradiol (E2) is shown on the right;

[0024] Figure 3 The figure shows the gender ratio of PL25 juvenile fed with 500, 1000, 2000 mg / kg MT and E2 for 40 days. Wherein: 17α-Methyltestosterone (MT) is shown on the left; Control group, 500 mg / kg group, 1000 mg / kg group and 2000 mg / kg group are shown in A-D in order; 17β-Estradiol (E2) is shown on the right; Control group, 500 mg / kg group, 1000 mg / kg group and 2000 mg / kg group are shown in A-D in order;

[0025] Figure 4Histological sections showing coexistence of testis and ovary; wherein: the upper shows 17α-Methyltestosterone (MT), from left to right: left shows atrophied oocytes, middle shows coexistence of testis and ovary observed in female shrimp after feeding with MT, right shows a large number of spermatogonia and a small number of sperm; the lower shows 17β-Estradiol (E2), from left to right: left shows a large number of spermatogonia, but no mature sperm observed, middle shows coexistence of testis and ovary observed in male shrimp after feeding with E2, right shows normally developed oocytes;

[0026] Figure 5 Flow chart showing the experimental procedure of feeding PL30 juvenile shrimp with 2000 mg / kg MT and E2; wherein: PL30, refers to the 30th day after metamorphosis; Sex reversal ratio, refers to the ratio of sex reversal;

[0027] Figure 6 Sex ratio after feeding PL30 juvenile shrimp with 2000 mg / kg MT and E2 for 50 days; wherein: left shows 17α-Methyltestosterone (MT); right shows 17β-Estradiol (E2). DETAILED DESCRIPTION

[0028] The present application discloses a method for sex reversal of crustaceans.

[0029] It should be understood that the expression "one or more of" includes individually each of the objects recited after the expression as well as various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0030] The use of the terms "including", "has", "having" or "contains" and grammatical variants thereof is generally understood either to mean "consists of" or "consists essentially of", unless otherwise understood from the context and usage. In this context, "consisting essentially of" means that the composition or method can include additional steps, elements, ingredients, components or species, but only if the additional steps, elements, ingredients, components or species do not materially affect the basic and novel characteristics of the composition or method.

[0031] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0032] The use of any and all examples, or exemplary language herein, for example, only the intention to better describe the application, and unless otherwise claimed, does not limit the scope of the application. Any language in this specification should not be interpreted as indicating any unclaimed element is essential to the practice of the application.

[0033] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are merely intended to convey general information as to the scope of the application. Consistent with the scope of the application, the numerical ranges and parameters in this specification are approximations, and are merely intended to convey general information as to the scope of the application. Consistent with the scope of the application, the numerical ranges and parameters in this specification are approximations and are merely intended to convey general information as to the scope of the application. For numerical ranges, the numerical values include the beginning and ending values, and all the other numbers and ranges between the beginning and ending values. Any numerical range is intended to include every narrower numerical range that falls within the broader numerical range. Also, any numerical range is intended to include every number falling within the range, inclusive of both endpoints and not inclusive of either endpoint. All percentages, ratios, and proportions herein are by weight, unless otherwise indicated. All percentages, ratios, and proportions are calculated based on the weight of the relevant substances, unless otherwise indicated.

[0034] The present application aims to determine the optimal sex reversal period and hormone concentration for different developmental stages of freshwater shrimp and to observe whether the sex-reversed individuals are fertile.

[0035] In Example 1 and Example 2 of the present application, the preparation method of the feed: the hormone is dissolved in 95% ethanol to prepare a stock solution with a concentration of 50 mg / mL, and dilute solutions of 5 mg / mL, 10 mg / mL and 20 mg / mL are prepared. The feed of different concentrations of ethanol is evenly sprayed (1 mL of ethanol per 10 g of feed), and a glass rod is stirred for at least 3 min. Then, it is placed under a ventilated laboratory engine cover and in a cool place for 15 minutes. The treated feed is placed in a 15 mL test tube and placed in a 0°C refrigerator to evaporate the remaining alcohol naturally. No hormone is added to the 95% ethanol, and the feed for the control group is prepared in the same way. The feed is stored in a 4°C refrigerator until feeding.

[0036] In Example 1 and Example 2 of the present application, the raw materials and reagents used can be purchased from the market.

[0037] The present application is further described below in conjunction with examples:

[0038] Example 1

[0039] Experimental method:

[0040] Normal development of female prawns were captured from the pond and cultured in a 500L water tank at room temperature (28±1°C) with oxygen concentration ≥6mg / L until the eggs were hatched. The fertilized eggs were cultured in the same environment until the second day and the 25th day after metamorphosis, and the experiment began. At the PL2 stage, 1000mg / kg of estrogen was fed, and at the PL25 stage, the concentration gradient of estrogen was set as 500mg / kg, 1000mg / kg and 2000mg / kg respectively. The feeding was twice a day (8.am and 8.pm) and the weight of each feeding was 2% of the body weight. The culture time was 40 days and the gender ratio was counted every 10 days. After metamorphosis, the development of gonads (testes, ovaries) needs 25 days, that is, to develop to PL25. It needs another 5-10 days to distinguish male and female through secondary sexual characteristics, so the gender ratio of the first three times cannot be observed in this experiment. Each concentration set three groups, each group of 200 juvenile prawns. When counting the gender ratio, 90 juvenile prawns were randomly selected from each group. Figure 1

[0041] Results:

[0042] 1. PL2 stage

[0043] MT feeding: the gender ratio (male: female) of the 1000mg / kg group was 1.4:1, which was significantly higher than that of the control group (as shown in Figure 2 left and Table 2);

[0044] E2 feeding: there was no significant difference between the experimental group and the control group (as shown in Figure 2 right and Table 1).

[0045] Table 1 Gender ratio of PL2 juvenile prawns fed with 1000mg / kg MT and E2 for 40 days

[0046]

[0047] Note: The data is expressed as mean ± standard error (n=90); different lowercase letters between the same column data indicate significant difference (P<0.05); different capital letters between the same column data indicate extremely significant difference (P<0.01).

[0048] 2. PL25 stage

[0049] MT feeding: compared with 500mg / kg and 1000mg / kg, the gender ratio of 2000mg / kg was higher and tended to be stable (as shown in Figure 3 left and Table 2). Histological observation found that there was a phenomenon of coexistence of testes and ovaries (as shown in Figure 4 above).

[0050] ​E2 feeding: All groups showed effect, and the sex ratio increased with the feeding time, but 2000 mg / kg showed the best effect (as shown in Figure 3 Table 2 and Table 3). Figure 4 Histological observation found the coexistence of testis-ovary (as shown in

[0051] Table 2 Sex ratio of PL25 juvenile shrimp fed with 500, 1000, 2000 mg / kg MT for 40 days

[0052]

[0053] Note: Data are expressed as mean ± standard error (n = 90); different lower case letters between the same column indicate significant difference (P < 0.05); different capital letters between the same column indicate extremely significant difference (P < 0.01).

[0054] Table 3 Sex ratio of PL25 juvenile shrimp fed with 500, 1000, 2000 mg / kg E2 for 40 days

[0055]

[0056]

[0057] Note: Data are expressed as mean ± standard error (n = 90); different lower case letters between the same column indicate significant difference (P < 0.05); different capital letters between the same column indicate extremely significant difference (P < 0.01).

[0058] Example 2

[0059] Experimental method:

[0060] The feed preparation and juvenile shrimp cultivation method are the same as Example 1. The fertilized eggs were cultivated to the 30th day after metamorphosis, and then the experiment was started by distinguishing male and female. Female shrimp were fed with 2000 mg / kg MT, and male shrimp were fed with 2000 mg / kg E2, twice a day (8 am and 8 pm), and the weight of each feeding (feeding period 25 days) was 2% of the body weight. The cultivation time was 50 days, and the sex ratio was counted every 25 days. (as shown in Figure 5 Table 4).

[0061] Experimental results:

[0062] PL30 period: MT feeding: 2000 mg / kg MT cannot reverse female shrimp to male shrimp, and no "pseudo-male shrimp" appeared in the experimental group (as shown in Figure 6 Table 4).

[0063] E2 feeding: 2000 mg / kg E2 successfully reversed male shrimps to female shrimps, about 20% of the experimental group appeared to carry eggs (such as Figure 6 Right and Table 4).

[0064] Table 4 Gender ratio of PL30 shrimps after 50 days of feeding with 2000 mg / kg MT and E2

[0065]

[0066] Note: Data are expressed as mean ± standard error (n = 90); different lower case letters between the same column data indicate significant difference (P <0.05); different capital letters between the same column data indicate extremely significant difference (P <0.01).

[0067] In summary, MT can induce PL2 and PL25 shrimps to be male, and E2 only has an effect on PL25 shrimps. From the results, MT is better. However, MT cannot reverse PL30 female shrimps, and E2 can reverse PL30 male shrimps.

[0068] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for sex reversal of a crustacean, characterized in that, The crustacean after metamorphic development is fed with feed added with steroid hormone, and is bred; the feeding includes: feeding the feed added with steroid hormone with a concentration of 1000 mg / kg on the second day after the metamorphic development or feeding the feed added with steroid hormone with a concentration of 2000 mg / kg on the 25th day after the metamorphic development; the steroid hormone includes: androgen; the androgen is: 17 alpha-methyltestosterone. When the feeding time is the second day or the 25th day after the metamorphic development, the breeding time is 40 days; the crustacean is: green shrimp.

2. The method of claim 1, wherein, The weight of the feeding is 2% of the body weight of the crustacean.

3. The method of claim 1 or 2, wherein, The temperature of the breeding is 27-29 DEG C, and the oxygen concentration is greater than or equal to 6 mg / L.

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