CN114216870A - Screening method and application of tussah breeding material - Google Patents
Screening method and application of tussah breeding material Download PDFInfo
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Abstract
The invention belongs to the technical field of silkworm breeding, in particular to a screening method of tussah breeding materials and application thereof, which comprises the steps of selecting different varieties of tussah breeding materials, obtaining semen generated in the mating process of male and female moths, carrying out acrosome enzyme activity measurement on the collected standby semen, counting the practical hatching rate of tussah eggs of each variety after the female moths of each variety of breeding materials lay eggs, establishing a regression equation according to the relation between the acrosome enzyme activity of each variety of sperms and the practical hatching rate of the tussah eggs, measuring the activity of the tussah sperm acrosome enzyme of different varieties of breeding materials in the production period of the 'two-in-one-out' of the tussah, predicting the practical hatching rate of the tussah eggs according to the established linear regression equation, screening the male breeding materials with high practical hatching rate of the tussah eggs, and selecting the male breeding materials in the mode, mating to obtain the egg with high hatching rate.
Description
Technical Field
The invention belongs to the technical field of silkworm breeding, and particularly relates to a screening method and application of tussah breeding materials.
Background
The tussah is also called as a wild silkworm, which originates from the Shandong province, Luzhongnan, China as the origin of the tussah industry, keeps the status of the first major country of the world tussah industry all the time, the total national output accounts for more than 90% of the total world output, the contribution rate of the Liaoning province is more than 60%, besides Liaoning, the main tussah production provinces such as Jilin, Heilongjiang and inner Mongolia also have rich tussah forest resources, and the total area of the tussah forest suitable for stocking the tussah in the 4 provinces reaches 346.3 ten thousand hm2But the practical application area is only 68.4 kilohm2The utilization rate is less than 20 percent, and the objective reason is that the provinces are in the north, the frost-free period is short, and the tussah can not be safely stocked in two seasons as a bivoltine tussah production area, so that the stocking enthusiasm of silkworm residents is low, and the tussah forest resource is left unused in a large area.
In order to solve the problems, the bivoltine first discharge of the tussah is realized by controlling the bivoltine tussah cocoon at low temperature, and the bivoltine tussah first discharge means that the bivoltine tussah cocoon is protected at low temperature in spring, the moth-developing period is delayed, and the bivoltine tussah spawns and hatches in spring and autumn, and is put in a stocking once a year. However, in practical application, the phenomenon that the hatching rate of the silkworm eggs is obviously reduced after the cocoons are stored at low temperature for a long time is found, the hatching rate of the silkworm eggs is a main character index of the vitality of the tussah silkworm eggs and is one of main economic indexes of the tussah silkworms, so that the obvious reduction of the practical hatching rate of the tussah silkworm eggs is also a main problem for limiting the development of a 'two-in-one-out' production mode, and the 'two-in-one-out' silkworm breeding scale only accounts for about 10% of the stocking area of a suitable area.
Therefore, the problem to be solved is urgently needed to ensure that the silkworm egg hatchability becomes 'two eggs one time release' production.
Disclosure of Invention
The invention aims to provide a screening method of a tussah breeding material and application thereof, and solves the technical problem that the hatching rate of silkworm eggs can be obviously reduced in a tussah two-part one-release culture method in the prior art.
The inventor of the application discovers that the practical hatchability of the tussah eggs is reduced in 'two chemical products one time' production through earlier stage tests, the reproductive capacity of male moths is reduced due to the fact that the activity of the sperm acrosome of the tussah silkworms is reduced, the two have a remarkable linear relation, a regression equation can be established according to the practical hatchability, meanwhile, the early stage tests also prove that the activity of the sperm acrosome of each tussah variety has remarkable difference, in conclusion, the practical hatchability of the tussah eggs can be predicted through measuring the activity of the sperm acrosome, and then male breeding materials with high activity of the sperm acrosome enzymes are screened out, namely male breeding materials with high practical hatchability of the tussah eggs.
The first aspect of the present invention is:
provides a method for screening tussah breeding materials, which comprises the following steps:
(1) selecting different varieties of tussah breeding materials, and respectively taking a plurality of male and female moths from the tussah breeding materials of each variety; in the process of mating male and female moths, dissecting the female moths, taking sperm beads out of mating capsules of the female moths, puncturing the sperm beads to absorb semen, placing the semen into an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out from each variety of tussah breeding materials;
calculating the quantity of sperms in each insect cell liquid culture medium, and calculating the quantity of sperms required to be extracted from the insect cell liquid culture medium as a group according to the quantity of sperms required to be tested by the sperm acrosome enzyme kit;
(2) according to the operation steps of the sperm acrosome enzyme kit, performing acrosome enzyme activity determination on each group of semen collected and reserved in the step (1) by utilizing an ultraviolet-visible spectrophotometer;
after female moths of various breeding materials lay eggs, a plurality of eggs laid by the female moths are taken and mixed into a group, and the practical hatchability is investigated;
(3) establishing a regression equation according to the obvious linear relation between the sperm acrosome enzyme activity X of each variety and the practical hatching rate Y of the tussah eggs; and (3) through a regression equation, the practical hatchability of the tussah eggs is deduced according to the activity of the acrosome enzyme of the tested sperms, and then the screening of mass breeding materials is carried out through testing the activity of the acrosome enzyme of the sperms.
Preferably, in the step (2), the sperm acrosome enzyme activity detection kit is a sperm acrosome enzyme activity detection kit produced by Tianjin Ruiyiki gold company;
preferably, in the step (3), the regression equation is established by using a sps data analysis software.
Preferably, in the step (1), at least 3 varieties of tussah breeding materials are selected, and at least 3 groups of semen are taken from each variety of tussah breeding material for dilution for later use;
in the step (2), after the female moths of each variety of tussah silkworm breeding material lay eggs, at least 3 groups of eggs are taken for the investigation of practical hatchability.
Preferably, in the step (1), the number of moths taken from each variety of tussah silkworm breeding material is determined according to the amount of the sperm required by the kit used for testing, and the amount of the sperm required by the test is ensured to be provided.
Preferably, in the step (1), the female moth is dissected within a period of 45min-1h15min when the male and female moths mate.
Preferably, in the step (1), the insect cell liquid medium is Grace's cell culture medium.
Preferably, in the step (1), the ratio is 10 × 106Individual sperm count diluted semen was drawn as a group.
Preferably, in the step (2), the UV-vis spectrophotometer is an shimadzu UV2600 UV-vis spectrophotometer.
The second aspect of the present invention is:
there is provided a method for screening a breeding material of Antheraea pernyi according to any one of the first aspect of the present invention, which is used for commercial breeding of Antheraea pernyi.
Compared with the prior art, the invention has the beneficial effects that: the screening method of the tussah breeding material is characterized in that in the production period of 'two generations and one release' of the tussah, the semen generated in the mating process of male and female moths is obtained by selecting a small amount of different varieties of tussah breeding materials, the acrosome enzyme activity of the collected standby semen is measured, after the female moths of various varieties of breeding materials lay eggs, the practical hatchability of the tussah eggs of various varieties is counted, a regression equation is established according to the obvious linear relationship between the acrosome enzyme activity of the sperms of various varieties and the practical hatchability of the tussah eggs, after the regression equation is established, the practical hatchability of the tussah eggs can be predicted only by testing the activity of the acrosome enzyme of the tussah sperms of different varieties of breeding materials, so that the male breeding material with high practical hatchability of the tussah eggs is screened out for the breeding of the 'two generations' tussah varieties, the mode is simple in operation and strong in practicability, and is suitable for screening the breeding materials of the breeding materials in batches and the breeding materials, the mating can obtain the eggs with high practical hatchability of the tussah eggs, can pertinently solve the difficult problem of low practical hatchability of the tussah eggs in the 'two-part one-discharge' production, and provides breeding materials for the 'two-part one-discharge' tussah variety breeding.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
A screening method of tussah breeding materials comprises the following steps:
step (1): selecting different varieties of tussah breeding materials, and respectively taking a plurality of male and female moths from the tussah breeding materials of each variety; in the process of mating male and female moths, dissecting the female moths, taking sperm beads out of mating capsules of the female moths, puncturing the sperm beads to absorb semen, placing the semen into an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out from each variety of tussah breeding materials; and calculating the quantity of sperms in the liquid culture medium of each insect cell, and calculating the quantity of sperms required to be extracted from the liquid culture medium of the insect cells as a group according to the quantity of sperms required to be tested by the sperm acrosome enzyme kit.
In this step, the selected breeding materials of tussah include "big 9", "phoenix long", "anti-big", "Liaowu silkworm 582", "black jade" and "Qinghuang No. 1", and the number of moths in the breeding materials of each variety is determined according to the amount of semen required by the reagent kit adopted in the test, and the amount of semen required by the test needs to be ensured.
The breeding materials of Dada 9, Fengjiu, Kangda, Liaosilkworm 582, Maoyu and Qinghuang No. 1 are bivoltine tussah silkworm varieties, all come from the breeding research laboratory of the national institute of silkworm industry in Liaoning province, all the supplied varieties are put in stock in autumn in the previous year, are stored in stock in a unified way, and are put out of stock in 5-26 days (the two-part one-time release seed production period) in 2021 year and heated until the moth comes out.
In addition, in the step, within the time period of 45min-1h15min after the male and female moths mate, the female moths are dissected, the semen beads are taken out and diluted by using an insect cell liquid culture medium with the same volume, wherein the insect cell liquid culture medium is Grace's cell culture medium, 3 groups of semen are taken from each variety of tussah silkworms and are diluted for later use, and each group is 10 multiplied by 106The amount of sperm was calculated for each sperm.
Step (2): according to the operation steps of the sperm acrosome enzyme kit, performing acrosome enzyme activity determination on each group of semen collected and reserved in the step (1) by utilizing an ultraviolet-visible spectrophotometer; after the female moths of various breeding materials lay eggs, a plurality of eggs laid by the female moths are taken and mixed into a group, and the practical hatchability is investigated.
In the step (2), the sperm acrosome enzyme activity detection kit is a sperm acrosome enzyme activity detection kit produced by Tianjin Ruiki company, the sperm acrosome enzyme activity detection kit of the company adopts a substrate enzyme method, and an ultraviolet visible spectrophotometer is an Shimadzu UV2600 ultraviolet visible spectrophotometer.
Sperm acrosome enzyme units are defined as: the amount of substrate hydrolysed at 24 ℃ by 1.0. mu. mol BAPNA/min is defined as 1IU acrosin activity.
The formula for calculating sperm acrosin is:
the following table 1 is a statistical table of sperm acrosome enzyme activities of different varieties of tussah silkworms in a biquai-lay seed production period, wherein 3 groups of semen are taken for measuring the tussah breeding material of each variety, as shown in the table 1, the sperm acrosome enzyme activities of the tussah breeding materials of various varieties have obvious difference, wherein the sperm acrosome enzyme activities measured by the tussah breeding material of the variety named after Fengjiu are obviously higher than those of the breeding materials of other varieties.
TABLE 1 Activity of anther acrosome spermatozoa acrosome enzymes of different varieties of tussah in 'two flowers and one breeding' seed production period
And (4) surface note: the test groups with different letters showed significant differences, including no significant difference with the same letters (P < 0.05).
After the female moths of various breeding materials lay eggs, taking 10 eggs laid by the female moths as a group for hatching, taking 3 groups of eggs for each variety, and counting the practical hatchability of the tussah eggs of each variety.
The following table 2 is the investigation result of the practical hatchability of different varieties of tussah eggs in the seed production period of the first two breeding batches, and it can be seen that the practical hatchability of the tussah eggs among the breeding materials of the varieties has significant difference, wherein the practical hatchability of the variety of the "Fengjiu" is significantly higher than that of other materials.
TABLE 2 survey results of practical hatchability of different varieties of tussah eggs in seed production period of two generations with one release
And (4) surface note: the test groups with different letters showed significant differences, including no significant difference with the same letters (P < 0.05).
And (3): establishing a regression equation according to the obvious linear relation between the sperm acrosome enzyme activity X of each variety and the practical hatching rate Y of the tussah eggs; through a regression equation, the practical hatchability of the tussah eggs can be deduced according to the activity of the acrosome enzyme of the test sperms, and then the screening of breeding materials in large batches is carried out only through the activity of the acrosome enzyme of the test sperms.
If different sperm acrosome enzyme activity detection kits are used in the step (2), and if the detection methods are different, the regression equation between the finally constructed sperm acrosome enzyme activity and the practical hatching rate of the tussah eggs is also different, and in the embodiment, the sperm acrosome enzyme activity detection kit produced by Tianjin Ruiyiki company is adopted, and in the step (3), the regression equation is that Y is 59.969+ 0.523X.
The following table 3 is a linear regression analysis table of the activity of the anther acrosome enzyme of the tussah silkworm and the practical hatchability of the tussah silkworm eggs in the seed production period of the first two-stage breeding, the table is obtained by performing linear regression analysis on the spss data analysis software, and as can be seen from the table 3, the linear regression analysis is performed on the statistical data in the table 1 and the table 2 to obtain a parameter 59.969 and a parameter 0.523, so that a linear regression equation Y is 59.969+ 0.523X.
In table 3, the value of the regression coefficient P is less than 0.05, which indicates that the practical hatchability of the top sperm enzyme activity X and the practical hatchability Y of the eggs of the oak silkworm are in a significant linear relationship, so the linear regression equation has a significant predictive effect on the practical hatchability of the eggs of the oak silkworm, and the practical hatchability of the eggs of the oak silkworm can be screened by measuring the sperm top sperm enzyme activity X.
TABLE 3 Linear regression analysis table of tussah sperm acrosome enzyme activity and tussah egg practical hatchability during seed production period
The screening method of the tussah breeding material is applied to the commercial breeding of tussah: in the production period of 'first two chemical breeding' of the tussah, the sperm acrosome enzyme activity detection kit produced by Tianjin Ruiki gold company is adopted to measure the sperm acrosome enzyme activity of the tussah of different varieties of breeding materials, and the practical hatchability of the silkworm eggs is predicted according to the linear regression equation Y which is 59.969+0.523X, so that the male breeding material with high practical hatchability of the silkworm eggs is screened out and used for breeding the 'first two chemical breeding' of the tussah varieties.
It should be noted that, in this document, terms such as "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A method for screening tussah breeding materials is characterized by comprising the following steps:
(1) selecting different varieties of tussah breeding materials, and respectively taking a plurality of male and female moths from the tussah breeding materials of each variety; in the process of mating male and female moths, dissecting the female moths, taking sperm beads out of mating capsules of the female moths, puncturing the sperm beads to absorb semen, placing the semen into an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out from each variety of tussah breeding materials;
calculating the quantity of sperms in each insect cell liquid culture medium, and calculating the quantity of sperms required to be extracted from the insect cell liquid culture medium as a group according to the quantity of sperms required to be tested by the sperm acrosome enzyme kit;
(2) according to the operation steps of the sperm acrosome enzyme kit, performing acrosome enzyme activity determination on each group of semen collected and reserved in the step (1) by utilizing an ultraviolet-visible spectrophotometer;
after female moths of various breeding materials lay eggs, a plurality of eggs laid by the female moths are taken and mixed into a group, and the practical hatchability is investigated;
(3) establishing a regression equation according to the obvious linear relation between the sperm acrosome enzyme activity X of each variety and the practical hatching rate Y of the tussah eggs; and (3) through a regression equation, the practical hatchability of the tussah eggs is deduced according to the activity of the acrosome enzyme of the tested sperms, and then the screening of mass breeding materials is carried out through testing the activity of the acrosome enzyme of the sperms.
2. The method for screening a breeding material of tussah silkworms according to claim 1, wherein:
in the step (2), the sperm acrosome enzyme activity detection kit is a sperm acrosome enzyme activity detection kit produced by Tianjin Ruiyiki Co.
3. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in step (3), the regression equation is established using the sps data analysis software.
4. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in the step (1), at least 3 varieties of tussah breeding materials are selected, and at least 3 groups of semen are taken from each variety of tussah breeding material for dilution for later use;
in the step (2), after the female moths of each variety of tussah silkworm breeding material lay eggs, at least 3 groups of eggs are taken for the investigation of practical hatchability.
5. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in the step (1), the number of moths in each variety of tussah breeding material is determined according to the amount of the sperm required by the kit adopted for testing, and the amount of the sperm required by the test needs to be ensured.
6. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in the step (1), the female moth is dissected within a time period of 45min-1h15min when the male and female moths mate.
7. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in the step (1), the insect cell liquid culture medium is Grace's cell culture medium.
8. The method for screening a breeding material of Antheraea pernyi as claimed in claim 1, wherein the step of screening is carried out byIn the following steps: in the step (1), according to
Individual sperm count diluted semen was drawn as a group.
9. The method for screening a breeding material of tussah silkworms according to claim 1, wherein: in the step (2), the UV-vis spectrophotometer is an shimadzu UV2600 UV-vis spectrophotometer.
10. The use of the method for screening Antheraea pernyi breeding material as claimed in any one of claims 1-9 in the commercial breeding of Antheraea pernyi.
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