CN113367097B

CN113367097B - Method for detecting continuous fertilization capability of hens

Info

Publication number: CN113367097B
Application number: CN202011412472.XA
Authority: CN
Inventors: 杨宁; 文超良; 孙从佼
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-12-03
Filing date: 2020-12-03
Publication date: 2022-11-18
Anticipated expiration: 2040-12-03
Also published as: CN113367097A

Abstract

The invention relates to the technical field of poultry genetic breeding, in particular to a method for detecting continuous fertilization capability of a hen. The method mainly comprises the following steps: evaluating and grouping the sperm quality of the cocks, diluting the mixed semen, artificially inseminating, collecting qualified hatching eggs, incubating and illuminating the eggs, and calculating the number of days (FDD) for continuous fertilization of the hens. The method has simple steps and easy implementation, fully considers the influence of the sperm quality and the egg quality of the cock on the fertilization of the hatching eggs, and focuses on the fertilization situation of the hatching eggs laid in the first few days after the insemination of the hens. FDD is used as an evaluation basis, and the continuous fertilization potential of the hens can be effectively reflected. The invention provides a new means and thought for accurately measuring the continuous fertilization ability of the hens, and has wide application value in the genetic improvement work of the continuous fertilization ability of the domestic hens.

Description

Method for detecting continuous fertilization capability of hens

Technical Field

The invention relates to the technical field of poultry genetic breeding, in particular to a method for detecting continuous fertilization capability of a hen.

Background

After natural mating or artificial insemination, hens can store sperm in the seminiferous glands in the oviduct and maintain the ability to lay fertilized eggs for a certain period of time, and this important physiological characteristic is called the Duration of fertilization (Duration of fertilization). Many studies have shown that there are significant individual differences in the sustained fertilization ability of hens, ranging from days to weeks.

In the modern large-scale and intensive chicken raising industry, an artificial insemination mode is generally adopted to ensure that the fertilization rate of hatching eggs is maintained at a higher level. The continuous fertilization ability of the hens is an important factor for determining the group fertilization level and the artificial insemination frequency, and the fertilization situation of the hatching eggs laid by the hens in the first few days after insemination is particularly important for actual production. Researches show that the fertilization rate of the hens reaches a peak gradually on the 2 nd day after insemination, and the fertilization rate rapidly decreases after 5 to 7 days, so that the interval between two insemination in production is generally 4 to 6 days. However, numerous studies have shown that hens may have 21-32 days between single mating or insemination and the last fertilized egg, with an average population of more than ten days. If we can improve the continuous fertilization ability of the population through genetic breeding, further improve or maintain the existing fertilization rate level, properly prolong the insemination interval, reduce the frequency of artificial insemination, undoubtedly greatly reduce the feeding amount of breeding cocks and obviously reduce the labor and feeding costs.

For genetic breeding, accurate phenotype determination is crucial to improving accuracy of breeding value estimation and accelerating breeding progress. The sustained fertilization ability of hens is a complex physiological characteristic involving sperm selection, storage, release, ascending to the site of fertilization, and sperm-egg binding. Research shows that after a single mating or insemination, a hen often has a plurality of non-fertilized eggs in the middle of the fertilized egg, and even the fertilized egg begins to be produced after a plurality of non-fertilized eggs are continuously produced. The semen storage performance of the hens can be measured by measuring the number of days between mating or semen deposition of the hens and the last fertilized egg, but the continuous fertilization capability of the hens cannot be accurately measured, and the method generally collects eggs for thirty days, wastes time and labor, and is difficult to popularize and apply comprehensively. Therefore, a method for measuring the continuous fertilization ability of the hens more quickly, conveniently and accurately is needed, and powerful technical support is provided for phenotypic determination of the continuous fertilization ability of the hens and genetic improvement of the continuous fertilization performance of the breeding hens.

Disclosure of Invention

In order to solve the technical problem, the invention provides a method for detecting the continuous fertilization capability of a hen.

Specifically, the technical scheme of the invention is as follows:

a method for detecting the continuous fertilization capability of a hen comprises the following steps:

(1) Evaluating and grouping the sperm quality of the cocks: collecting fresh semen of the alternative cocks, evaluating the quality of the semen of the cocks, and selecting the cocks with qualified semen quality;

(2) Diluting the mixed semen: collecting cock semen, mixing well, and diluting to obtain mixed diluted semen;

(3) Artificial insemination: carrying out artificial insemination on the hens to be evaluated for two consecutive days by using the mixed diluted semen;

(4) Collecting qualified hatching eggs: after two consecutive days of insemination, collecting qualified hatching eggs, and marking the blunt end of each egg to define egg collecting date and laying hens;

(5) And (5) hatching and lighting eggs: fumigating and sterilizing the collected qualified hatching eggs, placing the hatching eggs into a hatching room according to the egg collecting date, illuminating the eggs on the 7 th to 18 th days, and separately recording whether the hatching eggs are fertilized or not according to the egg collecting date;

(6) Calculating the number of days for continuous Fertilization (FDD) according to the egg control result;

the number of the continuous fertilization days is the number of the interval days between the last fertilized eggs and the number of the non-fertilized eggs produced in the interval time, which is subtracted from the number of the interval days between the two continuous artificial insemination days and the two non-fertilized eggs produced by the hen;

the invention finds that the method is favorable for accurately and effectively quantifying the continuous fertilization capability of the hen after the evaluation index of the continuous fertilization days is clarified.

Preferably, the alternative cock in the step (1) is subjected to semen collection training before the semen quality is assessed; the hen to be estimated in the step (3) is subjected to good insemination training and is stopped for more than 8 days before the artificial insemination for the continuous fertilization capability determination is carried out, so that the sperms in the seminal glands are emptied.

Preferably, in the step (1), the evaluation method of the quality of the cock semen is quick evaluation by a 200-400 times microscope visual method, and evaluation parameters comprise sperm density and motility.

Preferably, when the number of hens to be estimated is large, the number of the alternative cocks is increased, the cocks with qualified semen quality are grouped in the step (1), and 5-10 cocks are selected to be grouped according to the ejaculation amount of the cocks.

Preferably, the cock with excellent semen quality is selected in the step (1).

Further preferably, when evaluating the continuous fertilization ability of the whole group of hens, the number of the cocks used between the groups should be consistent and the semen quality similar.

Preferably, in the step (2), the dilution is specifically:

diluting the cock semen by using a diluent, wherein the diluent is one or more of a domestic chicken diluent, a phosphate buffer solution and physiological saline;

more preferably, the volume ratio of the diluent to the cock semen is 1:0.8 to 1.2, more preferably 1:1;

more preferably, the dilution is preheated to 30-38 ℃ before dilution.

Preferably, in the step (3), the artificial insemination is completed within 30 minutes after the cock insemination is completed.

Preferably, the insemination time is 15 to 18, more preferably, the hens have no hard-shell eggs in the uterus during insemination.

Preferably, the amount of the transferred fluid is 35 to 55. Mu.L, more preferably 50. Mu.L.

Preferably, in the step (3), the hens laying eggs within 40 minutes after the insemination is finished are supplemented; after the second insemination, the end egg is cleared.

Preferably, in the step (4), the qualified hatching eggs are collected for 12 to 18 days continuously after one day of insemination for two consecutive days according to the situation of the group laying rate.

Preferably, broken eggs, yin-yang eggs, double-yolk eggs and misshapen eggs are removed during collection.

In some embodiments, the manner in which laying hens are specified in step (4) is by labeling hen coop numbers; the egg collecting date and the laying hens can be simultaneously specified by marking the identification number, and the marking content is not specifically limited.

Preferably, the statistical result defaults to a missing value for hens that have less than one-third of the egg-trapping days in hatching.

The above-described preferred embodiments can be combined by one skilled in the art to provide preferred embodiments of the present invention.

Based on the scheme, the invention has the following beneficial effects:

(1) The method fully considers the influence of the sperm quality of the cock on the fertilization of the hatching egg, selects the cock with the middle-upper sperm quality to mix the sperm through the sperm quality microscopic examination, and avoids the interference of the sperm with higher or lower quality on the fertilization result. The sperm is treated in an equivalent dilution mode, the volume of the semen is increased, so that one group of cocks can be used for insemination of more hens, the damage to the semen caused by sperm metabolism is effectively reduced through dilution, and the sperms of the cocks in the same group are basically consistent in vitality when the insemination is started and ended. The artificial insemination mode is adopted, and the semen volume infused by each hen during insemination can be strictly controlled.

(2) The method quantifies the continuous fertilization capability of the hens, sets the statistical result of the hens with less number of eggs to be a deletion value, and avoids large errors caused by the fact that the number of the eggs is too small to be hatched to measure the result. Compared with the index of total Fertilized Egg Number (FEN), the FDD has weak correlation with the egg laying performance of the hens, and can effectively avoid the influence of the number of eggs which are hatched on a statistical result.

Wherein the total fertilized egg number is the total fertilized egg number of the hens to be estimated within the egg collecting days.

(3) In the egg collecting process, unqualified eggs are removed, and the influence on the evaluation of the continuous fertilization capability of the hens due to the quality of the eggs is reduced. And the egg collecting time is 12-18 days, the fertilization condition of the egg laid by the hen in the first 10 days after insemination is focused, compared with the Maximum Duration (MD) index, the method shortens the test period and reduces the labor cost. Compared with the effective fertilization days (ED) index of 'before heavy and light and after light', the invention considers the full-period fertilization condition within the egg collecting days.

Wherein the maximum duration days (MD) refers to the interval days from artificial insemination for two consecutive days to the last fertilized egg;

effective fertilization days (ED) are the number of days between two consecutive days of artificial insemination and the first non-fertilized egg;

"before heavy and after light" refers to fertilization in the first few days after excessive attention has been paid to insemination.

(4) The method has simple steps and easy implementation, fully considers the influence of the sperm quality and the egg quality of the cock on the fertilization of the hatching eggs and the egg number of the hen on the estimation result, and can effectively reflect the continuous fertilization potential of the hen.

(5) The invention provides a new means and thought for accurately determining and screening the continuous fertilization ability of the hens, and has wide application value in the genetic improvement work of the continuous fertilization ability of the hens.

Drawings

FIG. 1 is a technical route chart of a method for measuring the continuous fertilization ability of hens.

FIG. 2 is a graph showing the results of the egg-candling of some hens obtained in example 1 and statistics of the corresponding measures of the number of days of continuous Fertilization (FDD), the number of eggs in total Fertilization (FEN), the number of days of effective fertilization (ED), and the maximum number of days of continuous fertilization (MD).

FIG. 3 is a graph showing the correlation between each of the metrics of examples 1 and 2 and the number of eggs hatched.

FIG. 4 is the genetic association of the same scale between two weeks of age in examples 1 and 2.

FIG. 5 is a comparison of seed selection effect of two week-old integrated breeding values (weight ratio of 1:1) for each of the measures of examples 1 and 2 at 50% and 30% selective pressure.

FIG. 6 is a comparison of egg laying performance of the groups divided equally into two groups of high and low by the integrated breeding value of days for Fertilization (FDD) of example 1 and example 2.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1: method for detecting continuous fertilization capability of hens

In this example, 340 roosters with good semen collection reflex were selected from 34 weeks old pure line chickens, the number of hens was 2207, and the hens stopped semen deposition for 10 days before the experiment. The specific measurement process is as follows (the technical route diagram is shown in fig. 1):

1. evaluating the quality of cock semen: collecting cock semen in a 2.0mL centrifuge tube, sucking 25 μ L of fresh semen with a pipette, placing on a glass slide, covering with a cover glass, observing sperm motility and density under a 200-time microscope, and dividing semen quality into five grades of A, B, C, D and E, wherein the cock sperm with grade A has high density and good motility; the cock with the grade B has higher sperm density and better activity; the cock with the grade C has low sperm density or poor sperm motility; the cock with the grade D has low sperm density and poor vitality; cock sperm of grade E were all dead sperm or almost no sperm could be observed in the blinded area.

2. Grouping the cocks: 160 cocks with semen quality B are selected and grouped into one group according to the distance between the cocks and the cage position, and the groups are marked on the cage positions of the cocks in total of 32 groups. 16 groups of cocks before the first insemination and 16 groups of cocks after the second insemination.

3. Diluting mixed semen: after 14 pm on the third day after microscopic examination, the semen of each cock in the same group is collected in a semen collection cup, sucked into a 10mL centrifuge tube by a pipette gun or a rubber head dropper, and then sucked into an equal volume of preheated poultry diluent (38 ℃) for dilution and fully mixing.

4. Artificial insemination: after dilution and mixing are finished, a liquid transfer gun is used for insemination on the hens according to the cage position sequence, 120-160 hens are infused into each group of cocks, the insemination amount of each hen is 50 mu L, and the insemination mode is intravaginal insemination. All the insemination operations are completed within 30 minutes after each group of cocks finishes insemination, hens laying eggs within 40 minutes after insemination need to be supplemented, and hens which do not lay eggs need to register individual cage numbers and identification numbers.

5. Insemination in the next day: repeating

processes

3 and 4 for a second insemination of the entire group of hens, and removing the eggs from the end of the day after the entire group of hens has finished insemination.

6. Collecting qualified hatching eggs: hatching eggs are collected one day after two consecutive days of insemination, the number of qualified hatching eggs and non-qualified hatching eggs (broken eggs, yin-yang eggs, yolk eggs and malformed eggs) of each hen is recorded by an electronic scanner every day during the test period, the cage number of the hen is marked at the blunt end of the egg for the qualified hatching eggs, and the qualified hatching eggs in each day are separately placed according to the egg collecting date.

7. And (5) hatching and lighting eggs: and (3) fumigating and sterilizing all the collected qualified hatching eggs, incubating the hatching eggs together, sequentially photographing the hatching eggs on the 18 th day of incubation according to the egg collecting date, and sequentially registering the cage numbers marked on the non-fertilized eggs according to the egg collecting date.

8. And (3) calculating the balance indexes such as the number of continuous fertilization days (FDD): and (3) rejecting hens of which the number of insemination times is less than two, counting the Fertilization Duration Days (FDD), the total Fertilized Egg Number (FEN), the effective fertilization days (ED) and the maximum duration days (MD) of the hens in the test period by using R language for the rest hens according to egg laying and egg lighting records, defatting each index into a loss value for the hens of which the number of eggs to be hatched is less than 5, and finally, obtaining 2044 effective individual number of the hens.

The results of the measurement of the egg-exposure and fertilization-sustaining abilities of the partial hens obtained by the above-described procedure are shown in FIG. 2. The results of the population for continuous fertilization ability used in this example are shown in Table 1, and the results of the genetic parameter estimation are shown in Table 2.

TABLE 1.34 week old pure line chickens descriptive statistics of continuous fertilization ability

TABLE 2.34 evaluation results of genetic parameters of various measures of continuous fertilization ability of pure-line chickens

Note: diagonal lines are heritability and standard error, upper triangles are phenotype related, and lower triangles are inheritance related.

Example 2: method for detecting continuous fertilization capability of hens

In this example, 300 roosters with good semen collection reflex were selected from the 59-week-old pure line chickens, the number of hens was 2103, and the hens stopped to perform semen deposition for 10 days before the experiment. The specific measurement process is as follows:

1. evaluating the quality of cock semen: collecting cock semen in a 1.5 centrifuge tube, sucking 25 μ L of fresh semen with a pipette, placing on a glass slide, covering with a cover glass, observing sperm motility and density under a 200-time microscope, and dividing semen quality into five grades of A, B, C, D and E, wherein the cock sperm with grade A has high density and good motility; the cock with the grade B has higher sperm density and better activity; the cock with the grade C has low sperm density or poor sperm motility; the cock with the grade D has low sperm density and poor vitality; cock sperm of class E were all dead sperm or almost no sperm were observed in the blinded field.

2. Grouping the cocks: and selecting 140 cocks with semen quality B, grouping every five cocks into one group according to the distance between the cocks and the coop positions, wherein 28 groups are calculated, and grouping identification is made on each cock coop position. 14 groups of cocks before the first insemination and 14 groups of cocks after the second insemination.

3. Diluting the mixed semen: after 14 pm on the third day after microscopic examination, semen of each cock in the same group is collected in a semen collecting cup, sucked into a 10mL centrifuge tube by using a pipette gun or a rubber head dropper, and then diluted by sucking an equal volume of preheated poultry diluent (38 ℃) and fully mixed uniformly.

5. Insemination in the next day: repeating

processes

6. Collecting qualified hatching eggs: the method comprises the steps of collecting hatching eggs after two consecutive days of insemination, collecting the hatching eggs continuously for 15 days, recording the number of qualified hatching eggs and unqualified hatching eggs (broken eggs, yin-yang eggs, yolk eggs and malformed eggs) produced by each hen through an electronic scanner every day during the test period, identifying the cage number of the hen at the blunt end of the egg for the qualified hatching eggs, and separately placing the qualified hatching eggs every day according to the egg collecting date.

8. And (3) calculating the balance indexes such as the number of continuous fertilization days (FDD): and (3) rejecting hens of which the number of times of sperm injection is less than two, counting the number of continuous fertilization days (FDD), the number of total Fertilized Eggs (FEN), the number of effective fertilization days (ED) and the maximum duration days (MD) of the hens in the test period by utilizing an R language according to egg laying and egg lighting records of the remaining hens, and defatting each index to a deletion value for the hens of which the number of hatched eggs is less than 5. The final effective number of individuals was 1619 hens.

The results of the population for continuous fertilization ability used in this example are shown in Table 3, and the results of the genetic parameter estimation are shown in Table 4.

TABLE 3.59 descriptive statistics of the continuous fertilization ability of week-old inbred hens

TABLE 4.59 evaluation results of genetic parameters of various measurement indexes of continuous fertilization ability of pure line chickens of week age

The correlation coefficient between the metrics of example 1 and example 2 and the number of eggs hatched is shown in FIG. 3, which shows that the total Fertilized Egg Number (FEN) is highly correlated with the number of eggs hatched.

Example 1 and example 2 the genetic correlation of the same measures between two weeks of age is shown in figure 4, from which it can be seen that the effective fertilization days (ED) are less genetically correlated as measured in mid and late stages of egg production, and that the measures are not effective in measuring the sustained fertilization capacity of each egg production stage.

The comparison of the seed selection effects of the two week-old comprehensive breeding values (weight ratio of 1:1) of the measurement indexes of the examples 1 and 2 under the selection pressures of 50% and 30% is shown in fig. 5, and the seed selection effect of the maximum duration days (MD) is the worst; effective fertilization days (ED) can increase the fertilization rate of the first few days after insemination of the chicken flock, but cannot be maintained; and the breeding effect in the whole egg collecting period is better by taking the continuous fertilization days (FDD) as the breeding index.

The results of comparison of egg laying performance of the groups divided equally into two groups of high and low by the comprehensive breeding values of the number of days of continuous Fertilization (FDD) of example 1 and example 2 are shown in FIG. 6, the comprehensive breeding values are calculated by a weight ratio of Zhou Lingquan to 1:1 for two groups, and 1047 hens in each of the high and low groups. It can be seen from the figure that FDD is used as a breeding index, the phenotype values of FDD of two weeks are effectively improved, and the egg laying performance is not influenced.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for detecting the continuous fertilization capability of a hen is characterized by comprising the following steps:

(1) Evaluating and grouping the sperm quality of the cocks: collecting fresh semen of the alternative cocks, evaluating the quality of the semen of the cocks, and selecting and reserving the cocks with qualified semen quality;

supplementing and transporting the hens laying eggs within 40 minutes after the insemination; removing the eggs at the end of the day after the second insemination;

(4) Collecting qualified hatching eggs: after two consecutive days of insemination, starting to collect qualified hatching eggs one day after two consecutive days of insemination, continuously collecting the qualified hatching eggs for 12 to 18 days according to the egg laying rate condition of a group, and marking the blunt ends of the hatching eggs to determine the egg collecting date and the laying hens; removing broken eggs, yin-yang eggs, double-yolk eggs and malformed eggs during collection;

(5) And (5) hatching and lighting eggs: fumigating and sterilizing the collected qualified hatching eggs, placing the hatching eggs into a incubator for incubation in an open mode according to the egg collecting date, illuminating the eggs in 7 to 18 days, and recording whether the hatching eggs are fertilized or not according to the egg collecting date;

(6) Calculating the number of days for continuous fertilization according to the egg-lighting result;

for hens with less than one third of egg-collecting days after hatching, the statistical result is default to the deletion value.

2. The method according to claim 1, wherein the candidate cock in step (1) is subjected to semen collection training before the semen quality is assessed; the hen to be estimated in step (3) has undergone good insemination training and has withheld the insemination for more than 8 days before the determination of the continuous fertilization ability is carried out, so as to empty the sperms in the spermatic gland.

3. The method according to claim 1 or 2, wherein in the step (1), the evaluation method of the quality of the cock semen is quick evaluation by 200 to 400 times of microscope visual inspection, and the evaluation parameters comprise sperm density and motility.

4. The method according to claim 1, wherein when the number of hens to be estimated is large, the number of candidate cocks is increased, and the cocks with qualified semen quality are grouped in step (1), and 5 to 10 cocks are selected and grouped according to the ejaculation volume of the cocks.

5. The method of claim 4, wherein the number of roosters used between groups is consistent and semen quality is similar when evaluating the continuous fertilization ability of a whole group of hens.

6. The method according to claim 1, wherein in step (2), the dilution is specifically:

diluting cock semen with diluent, wherein the diluent is one or more of chicken diluent, phosphate buffer solution and physiological saline.

7. The method of claim 6, wherein the volume ratio of the diluent to the cock semen is 1:0.8 to 1.2.

8. The method according to claim 6, characterized in that the dilution is preheated to 30 to 38 ℃ before dilution.

9. The method according to claim 1, wherein in step (3), the artificial insemination is completed within 30 minutes after the cock is finished in insemination.

10. The method according to claim 9, wherein the insemination time is 15 to 00 to 18, and the hens have no hard-shelled eggs in the uterus during insemination.

11. The method of claim 9, wherein the amount of the input fluid is 35 to 55 μ L.