CN108840919B - Sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof - Google Patents
Sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof Download PDFInfo
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Abstract
The invention discloses a sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof. The invention provides a new sperm protein marker IZUMO2 closely related to breeding performance of breeding boars, the relative content of the sperm protein marker IZUMO2 is obviously and positively related to the breeding performance of the breeding boars, the sperm fertilization capability and the breeding performance of the breeding boars can be judged by detecting the relative content of the sperm protein IZUMO2, the breeding performance of the existing breeding boars can be accurately evaluated, breeding of the breeding boars with high breeding performance can be accurately carried out by utilizing the protein marker in production, the boars with poor breeding performance are eliminated, and the breeding production performance and the economic benefit of a pig farm can be improved. The method is convenient to use in production, and the semen protein marker IZUMO2 content can be detected, so that the sampling damage and stress of the breeding boar can not be caused.
Description
Technical Field
The invention relates to a sperm protein marker IZUMO2 for breeding boars with high reproductive performance, in particular to a method for judging the sperm fertilization capability and the reproductive performance of boars by detecting the relative content of sperm protein IZUMO2 so as to select boars with high reproductive performance from boar groups, wherein the relative content of sperm protein IZUMO2 of the boars is obviously and positively correlated with the reproductive performance of the boars.
Background
The improvement of livestock breeding is a core task of livestock science workers all the time, and therefore, a variety selection and breeding technology of a plurality of excellent livestock is established. The improvement of the reproductive performance of livestock is a main way for improving the production level and the economic benefit of the farm. However, the reproductive traits of livestock belong to the low heritability traits controlled by polygenes, and are difficult to improve by the existing genetic breeding technology.
The current breeding characteristics of pigs are related to the presence of genes such as Estrogen Receptor (ER), folliculotrophin β subunit (FSH- β), bone morphogenetic protein 15 (BMP 15), etc., which can be used for molecular marker-assisted selection of sows.
In the utilization of breeding boars, the semen is generally determined to be qualified by detecting the conventional semen quality (such as sperm density, sperm motility, teraticity, etc.) of boar semen, thereby determining whether the semen can be used for insemination. However, in actual production, the conventional quality of semen is the same, and the reproductive performance after insemination is greatly different. Therefore, routine quality inspection of semen does not accurately reflect the semen's fertility. The establishment of the evaluation index capable of reflecting the semen fertilization capability is beneficial to the evaluation of the semen fertilization capability and the evaluation of the breeding boar reproductive performance.
It is reported that although breeding boars are highly bred in western intensive pig farms, the breeding capacity of the boars is greatly different, and breeding performance of the boars is poor, and the parturition rate and litter size of the breeding boars are low. The data from the spanish artificial insemination center showed that when 10% of the breeder boars with ranked reproductive performance were removed, the litter size per sow could be increased by 2.23, which resulted in an increase in economic benefit of over 10000 euros per year in 1000-size farms (Roca et al 2015).
In recent years, with the development of proteomics and the application of gene knockout technology, more and more Sperm function-related proteins are discovered, which may be related to Sperm fertility and animal reproductive performance, such as Osteopontin (OPN), Epididymis Cysteine-Rich Secretory Protein 1 (CRISP 1) and Epididymis Protease inhibitor (EPPIN), Sperm binding Protein in Sperm serum (BSP), Sperm β -1, 4-galactose transferase (beta1, 4-galactosylsystem transferase, GalT), etc. Sperm Protein markers may help to identify fertile Sperm and sterility, but the related research is still in the primary stage, and the reliability of Sperm Protein markers in clinical tests still needs to be verified (Kwhon et al, see, Tan Sperm Protein in Sperm clearance, Sperm clearance N, Sperm secretion of Sperm cells in clinical tests, which may be considered as a Sperm fertility-related Protein component in Sperm fertility regulation of Sperm fertility and Sperm fertility of Sperm fertility, Sperm motility, and Sperm proliferation of Sperm cells in bovine Sperm cells, such as Sperm motility inhibition of Sperm motility, Sperm proliferation, Sperm secretion of Sperm Protein in Sperm cells in Sperm 23, Sperm secretion of Sperm Protein, Sperm Protein marker of Sperm 23, Sperm secretion of Sperm Protein, Sperm secretion of Sperm, Sperm secretion of Sperm, and Sperm proliferation of Sperm, thus, the Sperm secretion of Sperm Protein of Sperm, the Sperm of Sperm, the like the mouse, the Sperm of Sperm secretion of Sperm, the Sperm of Sperm Protein of Sperm, the Sperm of Sperm, the.
In 2015, Kwon et al examined protein expression in the post-capacitated sperm of high litter and low litter size white boars and discovered some differential proteins using two-dimensional electrophoresis and mass spectrometry techniques. Among them, ubiquinone cytochrome c reductase core protein II (ubiquitin-cytochrome c reductase core protein 2, UQCRC2) is up-regulated in the high litter size sperm, while ubiquinone cytochrome c reductase core protein I (ubiquitin-cytochrome c reductase core protein 1, UQCRC1), RAS protocarcinoma family member RAB2A (RAB2A member RASonogene family) and the like are highly expressed in the low litter size sperm. UQCRC2 is positively correlated with litter size, and RAB2A and UQCRC1 are negatively correlated with litter size. Biomarkers in these sperm have helped to discover high reproductive performance individual breeders in field trials.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a sperm protein marker IZUMO2 closely related to the reproductive performance of a breeding boar, and the breeding performance of the breeding boar can be judged by detecting the relative content of sperm protein IZUMO2 in the boar semen, so that the breeding boar with high reproductive performance can be bred from a breeding boar population.
Another object of the present invention is to provide the use of the sperm protein marker IZUMO 2.
The invention further aims to provide a method for breeding high-reproductive-performance boars by using the sperm protein marker IZUMO 2.
The purpose of the invention is realized by the following technical scheme:
the invention provides a sperm protein marker closely related to reproductive performance of breeding boars, and the sperm protein marker is a sperm protein marker IZUMO 2.
The invention also provides application of the sperm protein marker IZUMO2 in breeding boars with high reproductive performance. Wherein, the relative content of the sperm protein marker IZUMO2 is in obvious positive correlation with the reproductive performance of the breeding boar.
The invention also provides a method for breeding high-reproductive-performance breeding boars by utilizing the sperm protein marker IZUMO2, which comprises the following steps:
(1) according to the quantitative result of the sperm protein marker IZUMO2, various production performance indexes are combined, statistical analysis is carried out according to different selection proportions, and when the significance difference of the indexes among groups is determined, the relative content value of the sperm protein marker IZUMO2 is used as a reference threshold value for breeding boars with high reproductive performance in production;
(2) collecting semen of a breeding boar to be tested, and extracting a sperm protein sample;
(3) quantitatively detecting the content of the IZUMO2 protein marker in the sperm protein sample extracted in the step (2), and comparing the content with a reference threshold value to determine the reproductive performance of the boar to be detected, thereby breeding the boar with high reproductive performance and eliminating the boar with low reproductive performance.
The reference threshold value in the step (1) is that the breeding level is 68-86%, and the relative content range of the IZUMO2 protein is (11.25-14.02) × 10-8The breeding level is 68%, and the relative content range of the IZUMO2 protein is (12.36-14.02) × 10-8。
The preparation steps of the sperm protein sample in the step (2) are as follows:
preparing 30% and 60% percoll discontinuous density gradient solution into 15mL centrifuge tube, adding 3mL original semen, centrifuging at 20 deg.C for 20min at 400 × g, discarding supernatant, leaving only bottom sperm precipitate, centrifuging and washing sperm 3 times with DPBS, counting sperm, adjusting each tube 5-10 × 107Adding a protein extraction reagent according to the number of sperms, cracking for 0.5h, centrifuging at 14000 × g for 20min at 4 ℃, taking the supernatant to another centrifuge tube, adding 1.5 times of precooled acetone with the volume of-20 ℃, precipitating at-20 ℃ for 24h, centrifuging at 14000 × g for 20min at 4 ℃, discarding the supernatant, sucking the residual liquid, and storing at-80 ℃.
The quantitative determination described in step (3) employs the BCA (the bicinchoninic acid assay) and ELISA (the enzyme linked immunosorbent assay) methods.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a new sperm protein marker IZUMO2 closely related to breeding boar reproductive performance, which can realize more accurate evaluation on the reproductive performance of the existing breeding boars, can accurately carry out breeding of the breeding boars with high reproductive performance by utilizing the protein marker in production, eliminates the boars with poor reproductive performance, and is beneficial to improving the reproductive production performance and economic benefit of a pig farm.
(2) The sperm protein marker IZUMO2 provided by the invention is subjected to more systematic and strict test verification from the discovery of the protein marker to the verification of the protein function, so the protein marker is very accurate and reliable.
(3) The method is convenient to use in production, and the semen protein marker IZUMO2 content can be detected, so that the sampling damage and stress of the breeding boar can not be caused.
Drawings
Figure 1 is an ELISA validation of iTRAQ differential protein screening results.
FIG. 2 shows the expression of SPACA4 and IZUMO2 genes by RT-PCR, wherein β -actin is the reference gene, Lane M: 2000bp marker, Lane 1: adult boar testis, Lane 2: adult boar epididymis, Lane 3: adult boar vas deferens, Lane 4: 3 month-old boar testis, Lane 5: 3 month-old boar epididymis, and Lane 6: heart.
FIG. 3 shows Western blot detection of IZUMO2 protein; wherein, the lanes 1-3 are testis, epididymis and vas deferens of adult boar, the lanes 4-6 are testis, epididymis and vas deferens of 3-month-old boar, respectively, and the lane 7: heart, lane 8: sperm, lane 9: and (5) refining.
FIG. 4 is a comparison of the breeding performance of the boars of the high breeding group and the boars of the low breeding group with the IZUMO2 protein marker at 68% selection level, wherein the IZUMO2 sperm protein relative content of the boars of the low breeding group (7) is 6.35 × 10-8Very significantly lower than 13.19 × 10 of the high reproduction group (15 heads)-8(ii) a The mating childbirth rate, litter size and reproductive efficiency between the two groups were all significantly different.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The invention has two core technical links: establishing a boar reproductive performance related sperm protein marker IZUMO 2;
and (II) applying the IZUMO2 protein marker to realize the breeding of the breeding boars with high reproductive performance.
The method comprises the following specific steps:
establishment of sperm protein marker IZUMO2 related to boar reproductive performance
In order to establish the sperm protein marker IZUMO2 capable of reflecting the reproductive performance of the breeding boar and be used for breeding the high-reproductive-performance breeding boar, the research carries out the following work:
(1) according to the existing breeding production scores, representative breeding boars are scientifically and reasonably selected as research objects.
According to the records of breeding production performance of the breeding boars, important production indexes (breeding delivery rate, litter average litter size, litter average live litter size and breeding efficiency), semen conventional quality indexes (sperm motility and aberration rate) and in-vitro fertilization effect indexes (cleavage rate) are used for selecting more than 3 breeding boars with high breeding performance and low breeding performance from a breeding boar group as research objects.
Specifically, the hybridization production performance data of 745 long white breeding boars are collected from a large-scale original breeding pig farm, 10 breeding boars with high reproductive performance and 6 breeding boars with low reproductive performance are preliminarily screened out by establishing a mathematical model, and the specific reproductive performance conditions are shown in tables 1 and 2. And finally screening 3 breeding boars with high reproductive performance and 3 breeding boars with low reproductive performance as later-stage research objects through in vitro fertilization tests and research cost conditions, wherein specific test data are shown in tables 3 and 4. The results show that the routine index of semen of the selected two groups of boars has no difference, but the fertilization capability of the semen and the breeding performance index have obvious difference.
TABLE 1 comparison of the reproductive performance of boars of different reproductive performance breeds
| Group of | High reproductive performance | Low reproductive performance |
| Number of individuals (head) | 10 | 6 |
| Efficiency of reproduction*(head) | 8.58±0.88a | 6.93±0.80b |
| Parturition rate (%) | 79.23±6.07a | 72.41±6.47a |
| Total litter size of nest products (head) | 10.80±0.38a | 9.55±0.39b |
| Nest birth survival doll number (head) | 9.35±0.44a | 8.41±0.51b |
Note: the difference in the same row superscript letters indicates significant difference (P < 0.05).
*Propagation efficiency: means the number of effective offspring piglets produced by 1-birth sow of each mating boar.
TABLE 2 sperm motility and teratogenicity of boar semen of different reproductive performance
| Group of | Number of individuals | Sperm abnormality rate (%) | Sperm motility (%) |
| High |
10 | 8.3±1.2a | 88.0±1.0a |
| Low |
6 | 9.8±0.8a | 87.0±2.0a |
Note: sperm motility and teratogenicity were both averages of individuals within the group, with the same superscript letter indicating no significant difference (P > 0.05).
TABLE 3 comparison of IVF Effect of boar semen of different reproductive performance breeders
Note: the difference of the superscript letters among the groups indicates that the difference is significant, and p is less than 0.05.
TABLE 4 comparison of reproductive performance of boars of different groups of species finally screened
Note: the difference of the superscript letters among the groups indicates that the difference is significant, and p is less than 0.05.
(2) Collection of representative breeding boar semen and preparation of protein sample thereof
Semen of the 6-head Changbai boars is respectively collected by a conventional hand-held method, and the sperm density, activity and deformity rate of the boars are detected to be qualified. Sperm protein samples were prepared separately as follows:
preparation of sperm protein sample:
preparing 30% and 60% percoll discontinuous density gradient solution in 15mL centrifuge tube, adding 3mL semen, centrifuging at 20 deg.C for 20min at 400 × g, discarding supernatant, leaving only bottom sperm precipitate,washing sperm 3 times by using DPBS centrifugation, counting the sperm, adjusting each tube to 5-10 × 107Adding a protein extraction reagent according to the number of sperms, cracking for 0.5h, centrifuging at 14000 × g for 20min at 4 ℃, taking the supernatant to another centrifuge tube, adding 1.5 times of precooled acetone with the volume of-20 ℃, precipitating at-20 ℃ for 24h, centrifuging at 14000 × g for 20min at 4 ℃, discarding the supernatant, sucking the residual liquid, and storing at-80 ℃.
(3) Screening of boar sperm differential protein with different reproductive performance
The sperm protein samples were sent to the bioassay company for sperm-specific protein detection in two boar species using high throughput iTRAQ technology. The specific process is summarized as follows:
and carrying out reductive alkylation treatment on the extracted protein sample, and opening a disulfide bond so as to fully carry out enzymolysis on the protein in the subsequent steps. Protein concentration was determined by Brandford method. Protein sample quality was checked by SDS-PAGE electrophoresis (quality control). The same amount of protein Trypsin is taken from each sample for enzymolysis (quality control). Peptide fragments were labeled with iTRAQ reagent (quality control). The labeled peptide fragments were mixed in equal amounts and the mixed peptide fragments were pre-separated using strong cation exchange chromatography (SCX). Liquid phase tandem mass spectrometry (LC-MS/MS) analysis was performed.
Through quality control detection, all sperm proteins are qualified in quality and can be used for iTRAQ detection. There were 3 biological replicates and 2 technical replicates for each group of sperm protein assays. In relative quantification, the protein abundance ratio is close to 1 if the amount of the same protein does not vary significantly between the two samples. When the abundance ratio (namely, the difference multiple) of the protein reaches more than 1.2 and the p-value is smaller than 0.05 through statistical test, the protein is considered as the difference protein among different samples.
Through the iTRAQ detection, 42 sperm differential proteins (up-regulated 41 and down-regulated 1) exist in the high reproductive performance group and the low reproductive performance group. The difference protein GO is subjected to GO and KEGG pathway function enrichment analysis by using a David Bioinformatics Resources network database, and the result shows that the function of the difference protein GO is mainly concentrated on mitochondria, oxidized respiratory chain compound 1, electron transfer, metal combination and the like except disease related functions and signal channels, and the related main KEGG signal channel has oxidative phosphorylation, metabolism and the like.
Then, 2 of the sperm differential proteins (sperm acrosome associated protein 4 (SPACA 4)) and sperm membrane protein IZUMO2(IZUMO family member2)) are selected, and the relative content of the selected sperm acrosome associated protein and the selected sperm membrane protein is measured by using BCA (biochemical acid assay) and ELISA (enzyme-linked immunosorbent assay), so that the accuracy of the iTRAQ detection result is verified. The results are shown in fig. 1, and the variation trend of the difference among the 2 proteins is completely consistent with the iTRAQ results, which indicates that the reliability of the iTRAQ detection results is high.
IZUMO2 has two different forms:
form 1:
XM_021094779.1→XP_020950438.1 izumo sperm-egg fusion protein 2isoform X1
Conserved Domains(1)summary
pfam15005
Location:129→249
IZUMO；Izumo sperm-egg fusion,Ig domain-associated
form 2:
XM_021094780.1→XP_020950439.1 izumo sperm-egg fusion protein 2isoform X2
Conserved Domains(1)summary
pfam15005
Location:129→273
IZUMO；Izumo sperm-egg fusion,Ig domain-associated。
(4) establishment of sperm protein marker related to breeding boar reproductive performance
① correlation study of sperm IZUMO2 protein content and breeding boar reproductive performance
Semen of 22 service boars is collected from a stock pig farm in Guangdong province, and corresponding sperm protein samples are respectively prepared. The total protein concentration was determined by BCA method and the relative content of IZUMO2 protein was determined by ELISA method. Then, the correlation analysis of the relative content of IZUMO2 and the breeding performance of the breeding boars is carried out, and the result shows that the relative content of IZUMO2 sperm protein is in positive correlation (P <0.05) with the breeding delivery rate, the total litter size, the live litter size and the breeding efficiency of the breeding sows, and the correlation coefficients are 0.502, 0.569, 0.583 and 0.597 respectively (Table 5). The higher the relative content of the breeding boar sperm protein IZUMO2 is, the higher the reproductive performance is; the sperm protein IZUMO2 can be used as a protein marker reflecting the reproductive performance of breeding boars.
TABLE 5 correlation analysis of the relative content of IZUMO2 with the production performance of the Changbai boars
Note: the number of the long and white breeding boars is 22, and the number of the breeding sows is 1510; indicates significant at the 0.05 level (double-sided), indicates significant at the 0.01 level (double-sided).
② influence of sperm protein IZUMO2 on sperm motility and fertilization ability
The role of IZUMO2 protein in maintaining sperm motility and fertility was studied using antibody blocking. In the receptor fluid at a ratio of 1: the sperm motility was examined at different processing times with a fully automated sperm analyzer by adding IZUMO2 antibody at a dilution of 200, and the effect of protein antibody on sperm motility is shown in Table 6. There was no significant difference in the ratio of viable sperm between the control and IZUMO2 antibody groups at treatment times of 0min, 30min and 60min (P > 0.05). Indicating that the addition of IZUMO2 antibody does not have a serious effect on sperm motility.
TABLE 6 Effect of IZUMO2 antibody on sperm motility
Note: in the same column, the same superscript letter indicates no significant difference.
The effect of adding IZUMO2 antibody to the fertilization solution on the in vitro fertilization cleavage rate is shown in table 7, and the cleavage rate of the IZUMO2 antibody group is 39.88 ± 5.67%, which is significantly lower than that of the control group (P < 0.05). The results of this experiment show that the IZUMO2 protein on sperm has a significant positive effect on maintaining sperm's fertility. The finding of the result is helpful for explaining the relationship between the sperm differential protein and the reproductive capacity of the breeding boar.
TABLE 7 Effect of IZUMO2 antibody on cleavage Rate in vitro fertilization
| Grouping | Oocyte count (one) | Number of cleavage (one) | Cleavage Rate (%) |
| Control group | 313 | 184 | 58.29±12.84a |
| IZUMO2 antibody group | 304 | 122 | 39.88±5.67b |
Note: the difference of the superscript letters among the groups indicates that the difference is significant, and p is less than 0.05.
③ sperm protein IZUMO2 expression rule and cell location research
To further illustrate the way that sperm protein IZUMO2 affects sperm fertilization ability and breeding boar reproductive performance, we performed expression pattern and cell localization studies of the IZUMO2 protein.
Firstly, RT-PCR technology is utilized to detect the expression condition of IZUMO2 in RNA level in 3-month-old boar testis, epididymis and adult boar testis, epididymis, vas deferens and heart tissues. As a result, it was found that IZUMO2 was specifically expressed in the testis tissue of adult boars, but not in the testis, epididymis of 3-month-old boars, and epididymis, vas deferens and heart tissue of adult boars (FIG. 2).
The expression of the IZUMO2 protein in testis, epididymis and vas deferens of adult boars and 3-month-old testis, epididymis, vas deferens, heart, sperm and semen of boars is detected by western blot. As shown in FIG. 3, the IZUMO2 protein was expressed in both testis and sperm of adult boars, but not in epididymis, vas deferens and seminal plasma of adult boars; no expression of IZUMO2 protein was observed in 3-month-old boar testis, epididymis and vas deferens.
The experiment researches the expression condition of the IZUMO2 on the main reproductive organs and sperms of the boars, finds that the protein is distributed on both the testis and the sperms of the adult boars, and indicates that the protein possibly plays an important role in regulating and controlling the function or the fertilization capability of the sperms.
(5) Feasibility verification of using relative content of sperm protein marker IZUMO2 in breeding boar reproductive performance evaluation
In order to verify whether the sperm protein IZUMO2 can be used as a biomarker to breed high-reproductive-performance boars, 22 boars are selected from a certain original pig farm in Guangdong, sperm protein samples are respectively prepared, and the relative content of the IZUMO2 protein is measured by using a BCA (burst amplification of cell amplification) and an ELISA (enzyme-linked immunosorbent assay) method. And then, sequencing 22 long and white boars according to the breeding efficiency from high to low, selecting the first 11 boars as high breeding groups and the second 11 boars as low breeding groups, and checking whether the breeding production performance and the sperm protein content of IZUMO2 between the two groups are obviously different. The results are shown in Table 8: the content of the sperm protein of the IZUMO2 in the high breeding group is all obviously and extremely obviously higher than that in the low breeding group, and the delivery rate, the litter average litter size, the litter average live litter size and the breeding efficiency of the breeding boar of the high breeding group are all extremely and obviously higher than those of the low breeding group (P < 0.01). The relative content of the sperm protein IZUMO2 can reflect the reproductive performance of the breeding boar and can be used as a biomarker for screening the breeding boar with high reproductive performance.
TABLE 8 comparison of reproductive performance index and sperm protein content for boars of different reproductive performance
Note: in comparison with the same column, different upper case superscripts indicate very significant differences (P <0.01) and different lower case superscripts indicate significant differences (P < 0.05).
Screening threshold of (II) IZUMO2 protein marker and application effect thereof in breeding boar breeding
In order to establish a specific operation method for breeding the high-reproductive-performance boars by using sperm protein markers, 22 boars are respectively sorted according to the content of IZUMO2 protein, and production data of the boars are contrastively analyzed according to different breeding levels, so that the high-reproductive-performance boars are bred according to different breeding intensity requirements in production.
The breeding of high reproductive performance breeding boars according to the IZUMO2 protein marker has the following effects at different selection levels as shown in the following table 9. As can be seen from the table, the breeding of high reproductive performance breeding boars with the IZUMO2 sperm protein content has good effects, and when 86% of breeding levels (14% of breeding are eliminated), the IZUMO2 relative content of the reserved boars is (12.08 +/-0.83) × 10-8At 77% breeding level, the relative content of IZUMO2 of the reserved boar is (12.64 +/-0.83) × 10-8At 68% of breeding level, the relative content of IZUMO2 of the reserved boar is (13.19 +/-0.83) × 10-8The relative content of IZUMO2 of the reserved boar is (14.35 +/-0.91) × 10 at 50% of breeding level-8When the selective retention rate is between 68% and 86%, the breeding effect is good, and the relative content range of the IZUMO2 protein is (11.25-14.02) × 10-8The breeding level is 68%, and the relative content range of the IZUMO2 protein is (12.36-14.02) × 10-8(Table 9 and FIG. 4).
TABLE 9 Effect of breeding boars with IZUMO2 content at different selection levels
Note: at the same selection level, the superscript ns indicates no significant difference between groups, significant difference, and very significant difference.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. An application of sperm protein marker IZUMO2 closely related to breeding boar reproductive performance in breeding boars with high reproductive performance.
2. Use according to claim 1, characterized in that:
the relative content of the sperm protein marker IZUMO2 is in obvious positive correlation with the reproductive performance of the breeding boar.
3. A method for breeding boars with high reproductive performance by using sperm protein marker IZUMO2 is characterized by comprising the following steps:
(1) measuring the content of the sperm protein marker IZUMO2, grouping according to different selection proportions according to production performance indexes, and taking the relative content value of the sperm protein marker IZUMO2 as a reference threshold value for breeding high reproductive performance boars in production when the significance difference of each index between groups is determined; the production performance index is the mating childbirth rate, the litter average number born, the litter average number alive, the breeding efficiency, the sperm motility, the teratogenesis rate or the cleavage rate;
(2) collecting semen of a breeding boar to be tested, and extracting a sperm protein sample;
(3) quantitatively detecting the content of an IZUMO2 protein marker in the sperm protein sample extracted in the step (2), and comparing the content with a reference threshold value to determine the reproductive performance of the boar to be detected, so that the boar with high reproductive performance is bred, and the boar with low reproductive performance is eliminated;
the relative content of the sperm protein marker IZUMO2 is in obvious positive correlation with the reproductive performance of the breeding boar.
4. The method of claim 3, wherein:
the reference threshold value in the step (1) is the relative content of IZUMO2 protein when the selective retention rate is 68-86%, and the relative content range of IZUMO2 protein is (11.25-14.02) × 10-8。
5. The method according to claim 3 or 4, characterized in that:
the reference threshold value in the step (1) is the relative content of IZUMO2 protein when the selective retention rate is 68%, and the relative content range of the IZUMO2 protein is (12.36-14.02) × 10-8。
6. The method of claim 3, wherein:
the preparation steps of the sperm protein sample in the step (2) are as follows:
preparing 30% and 60% percoll discontinuous density gradient solution into 15mL centrifuge tube, adding 3mL original semen, centrifuging at 20 deg.C for 20min at 400 × g, discarding supernatant, leaving only bottom sperm precipitate, centrifuging and washing sperm 3 times with DPBS, counting sperm, adjusting each tube 5-10 × 107Adding a protein extraction reagent according to the number of sperms, cracking for 0.5h, centrifuging at 14000 × g for 20min at 4 ℃, taking the supernatant to another centrifuge tube, adding 1.5 times of precooled acetone with the volume of-20 ℃, precipitating at-20 ℃ for 24h, centrifuging at 14000 × g for 20min at 4 ℃, discarding the supernatant, sucking the residual liquid, and storing at-80 ℃.
7. The method of claim 3, wherein:
the quantitative detection in the step (3) adopts BCA and ELISA methods.
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