CN114216870B - Screening method of tussah breeding material and application thereof - Google Patents
Screening method of tussah breeding material and application thereof Download PDFInfo
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Abstract
The application belongs to the technical field of silkworm breeding, in particular to a screening method of tussah breeding materials and application thereof.
Description
Technical Field
The application belongs to the technical field of silkworm breeding, and particularly relates to a screening method of tussah breeding materials and application thereof.
Background
Tussah also called mountain silkworm, which originates in the Ruzhongnan area of Shandong province in China, china is used as the origin place of tussah industry, the first country of the world tussah industry is always kept, the total national yield is about more than 90% of the total world yield, the contribution rate of Liaoning province is more than 60%, besides Liaoning, the tussah main product provinces such as Jilin, heilongjiang and inner Mongolia also have rich tussah resources, and the total area of tussah suitable for raising tussah in the 4 provinces reaches 346.3 kilohm 2 But the practical application area is only 68.4 ten thousand hm 2 The utilization rate is insufficient20, the objective reason is that the north of the provincial area has short frost-free period, and the two-season tussah cannot be safely bred in the binarization tussah production area, so that the enthusiasm of silkworm breeding is low, and tussah forest resources are idle in a large area.
In order to solve the problems, the 'two-in-one putting' is realized by controlling the two-in-one tussah cocoons at a low temperature, and the 'two-in-one putting' of tussah is to protect the two-in-one tussah cocoons at a low temperature in spring, delay the moth-producing period, enable the tussah cocoons to spawn and hatch in spring and autumn and put the tussah in one 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, and the hatching rate of the silkworm eggs is a main character index of vitality of the tussah in the egg period and is also one of main economic indexes of the tussah, so that the obvious reduction of the practical hatching rate of the tussah eggs is also a main problem for limiting the development of a production mode of 'two-stage-one-stage', and the scale of 'two-stage-one-stage' silkworm breeding only accounts for about 10% of the stocking area of a proper area.
Therefore, the silkworm egg hatching rate is guaranteed to be a problem which needs to be solved in the 'two-in-one' production.
Disclosure of Invention
The application 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 is obviously reduced in a tussah 'two-in-one' breeding method in the prior art.
The inventor discovers through early-stage experiments that the practical hatching rate of tussah eggs in the production of 'two-stage one-stage' is obviously reduced due to the fact that the male breeding moth fertility is reduced caused by the fact that the activity of the top body enzyme of the tussah eggs is reduced, a regression equation can be established according to the practical hatching rate of the tussah eggs, meanwhile, the early-stage experiments also measure that the activity of the top body enzyme of the sperms among various tussah eggs is obviously different, in sum, the practical hatching rate of the tussah eggs can be predicted through measuring the activity of the top body enzyme of the sperms, and then male breeding materials with high activity of the top body enzyme of the sperms, namely male breeding materials with high practical hatching rate of the tussah eggs, are screened out.
The first aspect of the application is:
the screening method of the tussah breeding material 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; dissecting female moths in the mating process of male and female moths, taking out sperm beads from the mating bags of the female moths, puncturing the sperm beads to suck semen, placing the semen in an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out by the breeding materials of the tussah of each variety;
calculating the number of sperms in each insect cell liquid culture medium, and calculating the amount of semen required to be extracted from the insect cell liquid culture medium as a group according to the number of the test sperms required by the sperm acrosomal enzyme kit;
(2) According to the operation steps of the sperm acrosin kit, the ultraviolet-visible spectrophotometer is utilized to measure the activity of the acrosin of each group of semen which is collected and used in the step (1);
after female moths of various breeding materials spawn, mixing a plurality of eggs laid by the female moths into a group, and carrying out investigation on practical hatchability;
(3) Establishing a regression equation according to the obvious linear relation between the activity X of the acrosomal enzyme of each variety and the practical hatchability Y of the tussah eggs; the practical hatching rate of the tussah eggs is estimated according to the activity of the test sperm acrosin through a regression equation, and then the screening of mass breeding materials is carried out through the activity of the test sperm acrosin.
Preferably, in the step (2), the kit for detecting the activity of the acrosin is a kit for detecting the activity of the acrosin produced by Tianjin Ruijin company;
preferably, in the step (3), the regression equation is established by using spss 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 for diluting the tussah breeding materials of each variety for standby;
in the step (2), at least 3 groups of eggs are taken for investigation of practical hatchability after female moths of the tussah breeding materials of each variety lay eggs.
Preferably, in the step (1), the number of moths taken from the tussah breeding materials of each variety depends on the amount of semen required by the kit used for the test, and the amount of semen required by the test needs to be ensured.
Preferably, in the step (1), the female moth is dissected within a period of 45min to 1h and 15min in which mating of the male and female moths is performed.
Preferably, in the step (1), the insect cell liquid medium is Grace's cell medium.
Preferably, in the step (1), the method is carried out according to 10×10 6 Individual sperm amounts diluted semen was extracted as a group.
Preferably, in the step (2), the UV-vis spectrophotometer is an shimadzu UV2600 UV-vis spectrophotometer.
The second aspect of the application is:
there is provided a method of screening a tussah breeding material according to any one of the first aspects of the present application for use in commercial tussah breeding.
Compared with the prior art, the application has the beneficial effects that: according to the screening method of the tussah breeding material, during the production period of 'two-stage first-put' tussah, a small amount of different varieties of tussah breeding materials are selected, semen generated in the mating process of male and female moths is obtained, the collected semen is subjected to top enzyme activity measurement, after female moths of each variety of breeding materials lay eggs, practical hatchability of tussah eggs of each variety is counted, a regression equation is established according to a remarkable linear relation between the top enzyme activity of sperms of each variety 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 top enzyme activities 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 'two-stage first-put' tussah variety breeding.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
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; dissecting female moths in the mating process of male and female moths, taking out sperm beads from the mating bags of the female moths, puncturing the sperm beads to suck semen, placing the semen in an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out by the breeding materials of the tussah of each variety; the number of sperms in each insect cell liquid medium is calculated, and the amount of semen required to be extracted from the insect cell liquid medium is calculated as a group according to the number of sperms required to be tested by the sperm acrosomal enzyme kit.
In the step, the selected tussah breeding materials comprise tussah breeding materials of 'big 9', 'Fengjiu', 'anti-big', 'Liaojun', 'black jade', 'Qinghuang No. 1', and the number of the moths taken from the breeding materials of each variety is determined according to the semen amount required by the test adopted kit, so that the semen amount required by the test can be ensured.
The breeding materials of Dazhi 9, fengjiu, dazhao, liaosu 582, cutty jade and Qinghuang 1 are two-level tussah varieties, all come from the breeding research laboratory of Liaoning silkworm industry science research, all the tested varieties are uniformly bred in autumn of the previous year, are uniformly stored after being put in storage, and are uniformly discharged in a warehouse for 26 days of 2021 (the seed production period of two-level one discharge) until the moth is discharged.
In this step, the female moth is dissected and the sperm beads are taken out and used with an equal volume of insect cell liquid in a period of 45min to 1h and 15min for mating male and female mothsDiluting in Grace's cell culture medium, and diluting 3 groups of semen of tussah of each variety according to 10×10 6 Sperm count the amount of sperm.
Step (2): according to the operation steps of the sperm acrosin kit, the ultraviolet-visible spectrophotometer is utilized to measure the activity of the acrosin of each group of semen which is collected and used in the step (1); after female moths of various breeding materials spawn, a plurality of eggs laid by the female moths are mixed into a group, and the investigation of practical hatchability is carried out.
In the step (2), the sperm acrosin activity detection kit is a sperm acrosin activity detection kit produced by Tianjin Ruijin company, and the company sperm acrosin activity detection kit adopts a substrate enzyme method, and the ultraviolet visible spectrophotometer is an Shimadzu UV2600 ultraviolet visible spectrophotometer.
The definition of the sperm acrosin unit is: the amount of substrate hydrolyzed at 24℃to 1.0. Mu. Mol BAPNA/min was defined as 1IU of acroenzyme activity.
The calculation formula of the sperm acrosome enzyme is as follows:
the following table 1 is a statistical table of the activity of the acrosome enzyme of tussah of different varieties in the "two-stage one-stage" breeding period, 3 groups of semen are taken from the tussah breeding materials of each variety for measurement, as shown in table 1, the activity of the acrosome enzyme between the tussah breeding materials of each variety has a significant difference, wherein the activity of the acrosome enzyme measured by the tussah breeding materials of the "Fengjiu" variety is significantly higher than that of the other varieties.
TABLE 1- "two-stage one-stage" Oak silkworm sperm acrosome enzyme Activity in different species during seed production period
And (3) table notes: there was a significant difference between the test groups of different letters, including no significant difference (P < 0.05) for the same letter.
After female moths of various breeding materials spawn, taking 10 eggs laid by the moths as a group for hatching, taking 3 groups of eggs from each variety, and counting the practical hatching rate of tussah eggs of various varieties.
Table 2 below shows the results of the practical hatchability survey of tussah eggs of different varieties in the "two-stage-one-stage" seed production period, and shows that the practical hatchability of tussah eggs among the breeding materials of each variety has a significant difference, wherein the practical hatchability of the "Fengjiu" variety is significantly higher than that of other materials.
Table 2 results of investigation of practical hatchability of different varieties of tussah eggs in the "two-stage one-stage" seed production period
And (3) table notes: there was a significant difference between the test groups of different letters, including no significant difference (P < 0.05) for the same letter.
Step (3): establishing a regression equation according to the obvious linear relation between the activity X of the acrosomal enzyme of each variety and the practical hatchability Y of the tussah eggs; through a regression equation, the practical hatching rate of the tussah eggs can be deduced according to the test of the activity of the acrosomal enzyme, and then the screening of mass breeding materials is carried out only by the test of the activity of the acrosomal enzyme.
If different detection kits for the activity of the acrosin are used in the step (2), if the detection methods are different, the regression equation between the activity of the acrosin and the practical hatching rate of the tussah egg is also different, in this embodiment, the regression equation is y=59.969+0.523x in the step (3) by using the detection kit for the activity of the acrosin produced by the company Tianjin rayleigh.
Table 3 below is a linear regression analysis table of the activity of the acrosome enzyme of tussah sperm in the "two-stage-one-stage" production period and the practical hatchability of tussah eggs, which is obtained by linear regression analysis by the spss data analysis software, and as can be seen from table 3, the statistical data in table 1 and table 2 are subjected to linear regression analysis to obtain the parameters 59.969 and the parameters 0.523, thereby obtaining the linear regression equation y=59.969+0.523x.
In Table 3, the regression coefficient P value is less than 0.05, which shows that the acrozoospermia enzyme activity X and the practical incubation rate Y of tussah eggs are in a significant linear relation, so that the linear regression equation has a significant prediction effect on the practical incubation rate of tussah eggs, and tussah breeding materials with high practical incubation rates of tussah eggs can be screened by measuring the acrozoospermia enzyme activity X.
TABLE 3 Linear regression analysis Table of practical hatching rate of tussah silkworm egg and sperm acrosin Activity of "two-stage one-stage release" of tussah silkworm during seed production period
Application of screening method of tussah breeding material in tussah commercial breeding: in the production period of 'two-stage first-put' tussah, the sperm acrosin activity detection kit produced by Tianjin Ruijin corporation is adopted to measure the sperm acrosin activity of tussah of different varieties of breeding materials, and the practical incubation rate of the silkworm eggs is predicted according to a linear regression equation Y=59.969+0.523X, so that the male breeding materials with high practical incubation rate of the silkworm eggs are screened out and used for breeding 'two-stage first-put' 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 application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A screening method of 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; dissecting female moths in the mating process of male and female moths, taking out sperm beads from the mating bags of the female moths, puncturing the sperm beads to suck semen, placing the semen in an insect cell liquid culture medium with the same volume for dilution for later use, and respectively culturing the semen taken out by the breeding materials of the tussah of each variety;
calculating the number of sperms in each insect cell liquid culture medium, and calculating the amount of semen required to be extracted from the insect cell liquid culture medium as a group according to the number of the test sperms required by the sperm acrosomal enzyme kit;
(2) According to the operation steps of the sperm acrosin kit, the ultraviolet-visible spectrophotometer is utilized to measure the activity of the acrosin of each group of semen which is collected and used in the step (1);
after female moths of various breeding materials spawn, mixing a plurality of eggs laid by the female moths into a group, and carrying out investigation on practical hatchability;
(3) Establishing a regression equation according to the obvious linear relation between the activity X of the acrosomal enzyme of each variety and the practical hatchability Y of the tussah eggs; the practical hatching rate of the tussah eggs is estimated according to the activity of the test sperm acrosin through a regression equation, and then the screening of mass breeding materials is carried out through the activity of the test sperm acrosin.
2. The method for screening a tussah breeding material according to claim 1, wherein:
in the step (2), the kit for detecting the activity of the acrosin is a kit for detecting the activity of the acrosin produced by Tianjin Ruijin company.
3. The method for screening a tussah breeding material according to claim 1, wherein: in the step (3), a regression equation is established by using the spss data analysis software.
4. The method for screening a tussah breeding material according to claim 1, wherein: in the step (1), selecting at least 3 varieties of tussah breeding materials, and diluting at least 3 groups of semen for standby application;
in the step (2), at least 3 groups of eggs are taken for investigation of practical hatchability after female moths of the tussah breeding materials of each variety lay eggs.
5. The method for screening a tussah breeding material according to claim 1, wherein: in the step (1), the number of the moth of the tussah breeding materials of each variety is determined according to the semen amount required by the kit adopted in the test, and the semen amount required by the test is required to be ensured.
6. The method for screening a tussah breeding material according to claim 1, wherein: in the step (1), female moths are dissected within a period of 45min-1h and 15min, in which mating of male and female moths is performed.
7. The method for screening a tussah breeding material according to claim 1, wherein: in the step (1), the insect cell liquid medium is Grace's cell medium.
8. The method for screening a tussah breeding material according to claim 1, wherein: in the step (1), the following is followedIndividual sperm amounts diluted semen was extracted as a group.
9. The method for screening a tussah breeding material according to claim 1, wherein: in the step (2), the ultraviolet-visible spectrophotometer is an Shimadzu UV2600 ultraviolet-visible spectrophotometer.
10. Use of the screening method of the tussah breeding material according to any one of claims 1 to 9 in commercial tussah breeding.
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