CN108840919A - A kind of the sperm protein label IZUMO2 and its application closely related with herd boar reproductive performance - Google Patents
A kind of the sperm protein label IZUMO2 and its application closely related with herd boar reproductive performance Download PDFInfo
- Publication number
- CN108840919A CN108840919A CN201810614803.4A CN201810614803A CN108840919A CN 108840919 A CN108840919 A CN 108840919A CN 201810614803 A CN201810614803 A CN 201810614803A CN 108840919 A CN108840919 A CN 108840919A
- Authority
- CN
- China
- Prior art keywords
- sperm
- izumo2
- reproductive performance
- herd boar
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention discloses a kind of and closely related herd boar reproductive performance sperm protein label IZUMO2 and its application.The present invention provides a kind of new sperm proteins closely related with herd boar reproductive performance to mark IZUMO2, its relative amount and herd boar reproductive performance are positively correlated in significant, by the relative amount for detecting sperm protein IZUMO2, it just can determine whether the sperm fertility of herd boar and the height of reproductive performance, it may be implemented to carry out accurate evaluation to the reproductive performance of existing herd boar, it can use the breeding that the protein labeling accurately carries out high reproductive performance herd boar in production, reject the boar for eliminating reproductive performance difference, be conducive to improve the breeding Growth Results and economic benefit on pig farm.It is upper easy to use in production, IZUMO2 content is marked by detection spermatin, not will cause herd boar sampling damage and stress.
Description
Technical field
The present invention relates to a kind of sperm proteins of high reproductive performance herd boar breeding to mark IZUMO2, in particular to herd boar
The relative amount and herd boar reproductive performance of sperm protein IZUMO2 is positively correlated in significant, passes through detection sperm protein IZUMO2's
Relative amount, so that it may the sperm fertility of herd boar and the height of reproductive performance are judged, to select from herd boar group
Bring out the herd boar of high reproductive performance.
Background technique
The improved variety of livestock-raising is exactly the core missions of livestock technology worker all the time, is thereby established many excellent
The seed selection and breeding technology of breeding stock.And improving livestock reproduction performance is to improve the main way of farm's production level and economic benefit
Diameter.But the reproductive trait of domestic animal belongs to the low-heritability traits of controlled by multiple genes, is difficult to obtain by existing Biotechnology in Genetic Breeding
To improve.
Currently the relevant DNA molecular marker of pig reproductive trait has estrogen receptor (estrogen receptor, ER), promotees
Ovarian follicle element β subunit (follicle stimulating hormone-beta, FSH- β), 15 (bone of bone morphogenetic protein
Morphogenetic protein 15, BMP15) etc. genes, can be used for the molecular marker assisted selection of sow.In addition, also
Many SNP markers, for carrying out gene selects breeding.For herd boar breeding, current breeding objective usually only considers life
Character and Meat Quality are produced, without reproductive trait is included.Even if considering reproductive trait, current breeding technique such as base
Also it is difficult accurately in the traditional breeding techniques of Phenotypic Selection, Molecular Marker Assisted Selection Technology, full-length genome selection technique etc.
Select the herd boar of prolificacy.Therefore, causing the reproductive performance of current pig farm herd boar, to have height to have low, irregular, increases
The feeding cost of herd boar is added, and the excellent potential for hindering high reproductive performance herd boar cannot give full play to.So, if
A kind of selection method for boar reproductive performance can be established, so that the herd boar selected to molecular breeding in production is further
Carry out breeding？
In the utilization of herd boar, (such as sperm concentration, sperm are living for the general conventional semen quality by detection boar semen
Power, abnormal rate etc.) it inseminates to determine whether sperm is qualified, determining whether can be used for.But in actual production, there are sperm
Ordinary fruit quality is identical and breeds the huge phenomenon of achievement difference after insemination.Therefore, the Ordinary fruit quality inspection of sperm can not be quasi-
Really reflect the fertility of sperm.Establish the evaluation index for being able to reflect sperm fertility, it will help evaluate sperm
Fertility, and the height for assessing herd boar reproductive performance.
It is reported that, although herd boar is all the breeding that have passed through height, its reproductive capacity is still deposited in west Compact Develop
In larger difference, the boar of reproductive performance difference, the parturition rate and nest litter size of breeding are lower.Spain artificial insemination center's
Data show that after the 10% herd boar removal that reproductive performance is ranked behind, the every tire litter size of every sow can increase
2.23, this pig farm for allowing for 1000 scales increase every year 10000 Euros of economic benefit or more (Roca et al.,
2015)。
In recent years, with the application of the development of protein science and gene Knockout, more and more sperm functions are relevant
Protein is found, they may be related to sperm fertilizing ability and animal reproduction performance, as osteopontin (osteopontin,
OPN), epididymis rich in cysteine secretory protein 1 (Cysteine Rich Secretory Protein 1, CRISP1) and
Sperm binding protein in epididymis protease inhibitors (Epididymis Protease inhibitor, EPPIN), seminal plasma
(Binder of Sperm protein, BSP), sperm β-Isosorbide-5-Nitrae-galactosyl transferase (beta1,4-
Galactosyltransferase, GalT) etc..Sperm protein label can help to identify fertile sperm and sterile sperm, but related
Research still in the primary stage, and the reliability of sperm protein label still needed in clinical test verifying (Kwon et al.,
2015).It is reported that OPN level is related with its reproductive performance in Holstein sire seminal plasma；The secretion of people's epididymal epithelial cells
EPPIN can inhibit sperm motility；The adjustable sperm motility of seminal plasma protein B SP and capacitation process of bull seminal vesicle secretion,
It is considered as protein relevant to bull reproductive capacity；Mouse sperm protein Gal-9 T can be with zona pellucida 3 (zone
Pellucida 3, ZP3) it be combined with each other, and induce sperm acrosome reaction；The 14-kDa protein P14 (a in goat epididymis source
14-kDa protein) participate in acrosome film fusion.Therefore, the protein ingredient in sperm may be with the fertility of male
There are close relationship, related protein can be as the index of evaluation male sperm fertility and fertility.
2015, Kwon etc. had detected the albumen of sperm after the capacitation of high nest litter size and the long white race boar of low nest litter size
Expression has found some differential proteins using dielectrophoresis and mass-spectrometric technique.Wherein, sperm ubiquinone cytochrome c reduction
Enzyme core protein II (ubiquinol-cytochrome c reductase core protein 2, UQCRC2) is produced in high nest
It expresses and raises in the sperm of young number, and sperm ubiquinone Cytochrome c reductase core protein I (ubiquinol-cytochrome
C reductase core protein 1, UQCRC1), RAS original cancer family member RAB2A (RAB2A member RAS
Oncogene family) etc. the high expression in the sperm of low nest litter size.UQCRC2 is positively correlated with nest litter size, RAB2A and
UQCRC1 and nest litter size are negatively correlated.Biomarker in these sperms helps to find high reproductive performance in testing at the scene
Herd boar individual.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, it is bred the primary purpose of the present invention is that providing a kind of and herd boar
The closely related sperm protein of performance marks IZUMO2, can pass through sperm protein IZUMO2 relative amount in detection pig semen
It is how many to judge the height of herd boar reproductive performance, to select the herd boar of high reproductive performance from herd boar group.
Another object of the present invention is to provide the applications of above-mentioned sperm protein label IZUMO2.
It is public using the high reproductive performance kind of sperm protein label IZUMO2 breeding that a further object of the present invention is to provide a kind of
The method of pig.
The purpose of the invention is achieved by the following technical solution：
The present invention provides a kind of sperm protein label closely related with herd boar reproductive performance, the sperm protein mark
It is denoted as sperm protein label IZUMO2.
The present invention also provides a kind of application of sperm protein label IZUMO2 in the high reproductive performance herd boar of breeding.Its
In, sperm protein marks the relative amount of IZUMO2 and herd boar reproductive performance to be positively correlated in significant.
The present invention also provides a kind of methods using the sperm protein label high reproductive performance herd boar of IZUMO2 breeding, including
Following steps：
(1) quantitative result according to sperm protein label IZUMO2, in conjunction with various Growth Results indexs, according to different choosings
It is for statistical analysis to select ratio, for each index there are when significant difference, sperm protein label IZUMO2's is opposite between determining group
Content value, as the reference threshold for being used for the high reproductive performance herd boar of breeding in production；
(2) sperm for acquiring breeding herd boar to be measured, extracts sperm protein sample；
(3) quantitative detection step (2) extract sperm protein sample in IZUMO2 protein labeling content, by with reference
The comparison of threshold value determines the height of boar reproductive performance to be measured, to select high reproductive performance herd boar, eliminates low breeding
Performance herd boar.
Reference threshold described in step (1) is that breeding level is 68%~86%, at this time IZUMO2 albumen relative amount
Range is (11.25~14.02) × 10-8；Further preferably breeding level is 68%, at this time IZUMO2 albumen relative amount model
Enclosing is (12.36~14.02) × 10-8。
The preparation step of sperm protein sample described in step (2) is as follows：
The former essence of 3mL is added in 15mL centrifuge tube in the percoll discontinuous density gradient liquid of preparation 30% and 60% on it
Liquid, 400 × g is centrifuged 20min at 20 DEG C, discards supernatant, portion's Sperm pellets of only keeping on file, with DPBS centrifuge washing sperm 3 times, essence
Sub-count adjusts every pipe 5~10 × 107A sperm is added Protein Extraction Reagent according to sperm quantity, 0.5h is cracked, under the conditions of 4 DEG C
14000 × g is centrifuged 20min, takes supernatant into another centrifuge tube, and -20 DEG C of pre- cold acetones of 1.5 times of volumes, -20 DEG C of sinkings are added
It forms sediment for 24 hours, then 14000 × g is centrifuged 20min at 4 DEG C, abandons supernatant, blot raffinate, saved in -80 DEG C.
Quantitative detection described in step (3) use using BCA (the bicinchonininc acid assay) and
ELISA (the enzyme linked immunosorbent assay) method.
The present invention has the following advantages and effects with respect to the prior art：
(1) the present invention provides a kind of new sperm proteins closely related with herd boar reproductive performance to mark IZUMO2,
It may be implemented to carry out accurate evaluation to the reproductive performance of existing herd boar, it is accurate to can use the protein labeling in production
Ground carries out the breeding of high reproductive performance herd boar, rejects the boar for eliminating reproductive performance difference, and the breeding for being conducive to improve pig farm is raw
Produce achievement and economic benefit.
(2) sperm protein provided by the invention marks IZUMO2, from the discovery of protein labeling to the verifying of protein function,
The stringent verification experimental verification compared with system is carried out, so the protein labeling is very accurate and reliable.
(3) upper easy to use in production, IZUMO2 content is marked by detection spermatin, not will cause herd boar
Sampling damages and stress.
Detailed description of the invention
Fig. 1 is the ELISA verifying of iTRAQ differential protein the selection result.
Fig. 2 is SPACA4 and IZUMO2 gene expression RT-PCR detection；Wherein, β-actin is reference gene；Swimming lane M：
2000bp marker, swimming lane 1：Adult male pig testis, swimming lane 2：Adult male pig epididymis, swimming lane 3：Adult male pig vas deferens, swimming lane
4：3 monthly age Testis of Boar Pig, swimming lane 5：3 monthly age boar epididymises, swimming lane 6：The heart.
Fig. 3 is IZUMO2 albumen Western blot detection；Wherein, swimming lane 1-3 be respectively adult male pig testis, epididymis and
Vas deferens, swimming lane 4-6 are respectively 3 monthly age Testis of Boar Pig, epididymis and vas deferens, swimming lane 7：The heart, swimming lane 8：Sperm, swimming lane 9：Essence
Clearly.
Fig. 4 is the breeding achievement for selecting and remain numerous group of the height and low numerous group of herd boar under level 68% with IZUMO2 protein labeling
Compare；The IZUMO2 sperm protein relative amount average out to 6.35 × 10 of numerous group low (7) herd boar-8, extremely significant to be lower than Gao Fan
The 13.19 × 10 of group (15)-8；Breeding parturition rate, number born per litter, nest young number living and reproductive efficiency between two groups all have
Significant difference.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
Two core technology links of the invention：(1) herd boar reproductive performance correlation sperm protein marks IZUMO2 really
It is vertical；
(2) breeding of high reproductive performance herd boar is realized using IZUMO2 protein labeling.
Specific step is as follows：
(1) establishment of herd boar reproductive performance correlation sperm protein label IZUMO2
In order to establish the spermatin label IZUMO2 for being able to reflect herd boar reproductive performance, for high reproductive performance kind public affairs
The breeding of pig, this research have carried out following work：
(1) according to having breeding Growth Results, scientific and reasonable selects representativeness herd boar as research object.
According to herd boar breeding Growth Results record, while with important production target (breeding parturition rate, number born per litter,
Nest young number living and reproductive efficiency), the Semen routione index of quality (sperm motility, abnormal rate) and effectiveness indicator (ovum in vitro fertilization
Split rate) high reproductive performance and each 3 of low reproductive performance herd boar or more are selected from herd boar group, as research object.
Specifically, the breeding Growth Results data of 745 long white race boar are had collected from scale original seed pig farm, by building
Vertical mathematical model preliminary screening goes out the herd boar of 10 head height reproductive performances and 6 low reproductive performances, and specific reproductive performance situation is shown in
Tables 1 and 2.3 head height reproductive performances and 3 low breedings are selected by test in vitro fertilization and research cost situation, finishing screen again
Performance herd boar is shown in Table 3 and table 4 as later period research object, specific test data.The results show that selected two groups of kinds are public
Pig, Semen routione index does not have difference, but has significant difference between the fertility of sperm and breeding reproductive performance.
The comparison of 1 different reproductive performance herd boar of table breeding achievement
| Group | High reproductive performance | Low reproductive performance |
| Number of individuals (head) | 10 | 6 |
| Reproductive efficiency*(head) | 8.58±0.88a | 6.93±0.80b |
| Parturition rate (%) | 79.23±6.07a | 72.41±6.47a |
| Nest produces total young number (head) | 10.80±0.38a | 9.55±0.39b |
| The number of live birth (head) | 9.35±0.44a | 8.41±0.51b |
Note：Superscript letters difference of going together indicates significant difference (P<0.05).
*Reproductive efficiency：Refer to effective offspring piglet number that herd boar 1 tire sow of every breeding is able to produce out.
The sperm motility of 2 different reproductive performance herd boar sperm of table is compared with abnormal rate
| Group | Number of individuals | Rate of teratosperm (%) | Sperm motility (%) |
| High reproductive performance | 10 | 8.3±1.2a | 88.0±1.0a |
| Low reproductive performance | 6 | 9.8±0.8a | 87.0±2.0a |
Note：Sperm motility and abnormal rate are the average value of individual in group, and identical Superscript letters indicate the not significant (P of difference>
0.05)。
The IVF effect of 3 different reproductive performance herd boar sperm of table compares
Note：Superscript letters difference indicates significant difference, p between group<0.05.
The comparison for the different group herd boars breeding achievement that table 4 finally screens
Note：Superscript letters difference indicates significant difference, p between group<0.05.
(2) acquisition of representative herd boar sperm and its preparation of protein sample
The sperm for acquiring above-mentioned 6 long white race boars respectively using conventional hand grip detects its sperm concentration, vigor and abnormal
Form quotient is qualified.Sperm protein sample is prepared respectively, and method is as follows：
The preparation of sperm protein sample：
The former essence of 3mL is added in 15mL centrifuge tube in the percoll discontinuous density gradient liquid of preparation 30% and 60% on it
Liquid, 400 × g is centrifuged 20min at 20 DEG C, discards supernatant, portion's Sperm pellets of only keeping on file, with DPBS centrifuge washing sperm 3 times, essence
Sub-count adjusts every pipe 5~10 × 107A sperm is added Protein Extraction Reagent according to sperm quantity, 0.5h is cracked, under the conditions of 4 DEG C
14000 × g is centrifuged 20min, takes supernatant into another centrifuge tube, and -20 DEG C of pre- cold acetones of 1.5 times of volumes, -20 DEG C of sinkings are added
It forms sediment for 24 hours, then 14000 × g is centrifuged 20min at 4 DEG C, abandons supernatant, blot raffinate, saved in -80 DEG C.
(3) screening of different reproductive performance herd boar sperm differential protein
Sperm protein sample is sent to biological detection company, the essence of two groups of herd boars is detected using high-throughput iTRAQ technology
Sub- differential protein.Detailed process is summarized as follows：
Reductive alkylation processing is carried out to the protein sample after extraction, opened disulfide bond sufficiently digests egg so as to subsequent step
It is white.The concentration mensuration of albumen is carried out with Brandford method.With SDS-PAGE electrophoresis detection protein sample quality (Quality Control).Each
Sample takes equal protein Trypsin to digest (Quality Control).Peptide fragment (Quality Control) is marked with iTRAQ reagent.Peptide fragment after label is carried out
Mixed in equal amounts carries out pre-separation using strong cation exchange chromatography (SCX) to mixed peptide fragment.Carry out liquid phase tandem mass spectrum
(LC-MS/MS) it analyzes.
It is detected by Quality Control, all sperm proteins are up-to-standard, can be utilized for iTRAQ detection.Every group of sperm protein inspection
Survey has 3 biology to repeat, and 2 technologies repeat.In relative quantification, if the amount of the same albumen does not have in two sample rooms
Significant changes, then its protein abundance ratio is close to 1.When the abundance ratio (i.e. fold differences) of albumen reaches 1.2 or more, and warp
When crossing statistical check its p-value value less than 0.05, the albumen is regarded as the differential protein of different sample rooms.
Detected through iTRAQ, the sperm differential protein of high reproductive performance group and low reproductive performance group have 42 (up-regulation 41,
Lower 1).Using David Bioinformatics Resources network data base to above-mentioned differential protein carry out GO and
The enrichment analysis of KEGG pathway function, as a result, it has been found that, in addition to disease correlation function and signal path, differential protein GO function master
Mitochondria, oxidation-respiration chain compound 1, electron transmission, metal bonding etc. are concentrated on, the main KEGG signal path being related to has
Oxidative phosphorylation and metabolism etc..
Then, we choose 2 ((sperm acrosome of perforatorium GAP-associated protein GAP 4 from sperm differential protein
Associated 4, SPACA4), Researches on Sperm Membrane Proteins IZUMO2 (IZUMO family member2)), utilize BCA (the
Bicinchonininc acid assay) and ELISA method carry out relative amount measurement to it, verifying iTRAQ testing result
Accuracy.As a result as shown in Figure 1, the group difference variation tendency and iTRAQ result of 2 kinds of albumen are completely the same, show that iTRAQ is examined
The reliability for surveying result is higher.
IZUMO2 has two different forms：
Form 1：
XM_021094779.1→XP_020950438.1 izumo sperm-egg fusion protein 2
isoform 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 2
isoform X2
Conserved Domains(1)summary
pfam15005
Location:129→273
IZUMO；Izumo sperm-egg fusion,Ig domain-associated.
(4) establishment of herd boar reproductive performance correlation sperm protein label
1. sperm IZUMO2 protein content and herd boar reproductive performance correlation research
The sperm that 22 in-service herd boars are acquired from Guangdong Province original seed pig farm, is prepared for corresponding sperm protein sample respectively
Product.Total protein concentration measurement is carried out using BCA method, the relative amount of IZUMO2 albumen is determined using ELISA method.Then it carries out
The correlation analysis of IZUMO2 relative amount and herd boar breeding achievement, as a result, it has been found that, IZUMO2 sperm protein relative amount
It is in significantly positive correlation (P with the breeding parturition rate for matching sow, the total young number of nest production, the number of live birth, reproductive efficiency is joined<0.05),
Related coefficient is respectively 0.502,0.569,0.583 and 0.597 (table 5).Show herd boar sperm protein IZUMO2 relative amount
Higher, reproductive performance is higher；Sperm protein IZUMO2 can be used as the protein labeling of reflection herd boar reproductive performance.
The correlation analysis of table 5 IZUMO2 relative amount and long white race boar Growth Results
Note：Long white race boar n=22 head, with number 1510, broad sow head；* it indicates to show on 0.05 horizontal (bilateral)
It writes, * * indicates significant on 0.01 horizontal (bilateral).
2. influence of the sperm protein IZUMO2 to sperm motility and fertility
Effect in sperm motility and fertility is being maintained using antibody closed technique study IZUMO2 albumen.?
By in sperm with 1：200 dilution ratios add IZUMO2 antibody, detect essence with computer assisted sperm analysis instrument in the different disposal time
Sub- vigor, influence of the protein antibodies to sperm motility are as shown in table 6.When handling the time is 0min, 30min and 60min, control
The motile sperm ratio with IZUMO2 antibody group is organized without significant difference (P>0.05).Illustrate that addition IZUMO2 antibody will not be to essence
Sub- vigor generation seriously affects.
Influence of the 6 IZUMO2 antibody of table to sperm motility
Note：In same row, the identical expression difference of Superscript letters is not significant.
As shown in table 7, IZUMO2 antibody group is being influenced on spilting of an egg rate in vitro fertilization by IZUMO2 antibody is added in sperm
Cleavage rates be 39.88 ± 5.67%, substantially less than control group (P<0.05).This test result shows the IZUMO2 on sperm
Albumen is for maintaining the fertility of sperm to have significant positive effect.The discovery of the result helps to illustrate sperm difference
Relationship between albumen and herd boar fertility.
Influence of the 7 IZUMO2 antibody of table to spilting of an egg rate in vitro fertilization
| Grouping | Oocyte number (piece) | Spilting of an egg number (piece) | Cleavage rates (%) |
| Control group | 313 | 184 | 58.29±12.84a |
| IZUMO2 antibody group | 304 | 122 | 39.88±5.67b |
Note：Superscript letters difference indicates significant difference, p between group<0.05.
3. the expression rule of sperm protein IZUMO2 and cellular localization research
The approach of sperm fertility and herd boar reproductive performance is influenced in order to further illustrate sperm protein IZUMO2, I
Carried out IZUMO2 albumen expression pattern and cellular localization research.
3 monthly age Testis of Boar Pig, epididymis and adult male pig testis, epididymis, vas deferens are had detected first with RT-PCR technology
With IZUMO2 in heart tissue rna level expression.As a result, it has been found that IZUMO2 is in the testis tissue of adult male pig
Specifically expressing is showed no expression (figure in the testis of 3 monthly age boars, the epididymis of epididymis and adult male pig, vas deferens and heart tissue
2)。
Then to IZUMO2 albumen, using western blot have detected its adult male pig testis, epididymis, vas deferens with
And the expression in 3 monthly age Testis of Boar Pig, epididymis, vas deferens, heart, sperm and seminal plasma.As a result as shown in figure 3, IZUMO2
Albumen has expression in adult male pig testis and sperm, and expression is had no in adult male pig epididymis, vas deferens and seminal plasma；?
The expression of IZUMO2 albumen is had no in 3 monthly age Testis of Boar Pig, epididymis and vas deferens.
This part experimental study expression of the IZUMO2 in boar Main Reproductive Organs and sperm, finds the albumen
It is distributed on adult male pig testis and sperm, implies that above-mentioned albumen may be in terms of regulation sperm function or fertility
Play a significant role.
(5) feasibility verifying of the sperm protein label IZUMO2 relative amount for the evaluation of herd boar reproductive performance
Can be as the high reproductive performance herd boar of biomarker breeding in order to verify sperm protein IZUMO2, we are from Guangdong
22 herd boars are selected in certain original seed pig farm, prepare its sperm protein sample respectively, and carry out using BCA and ELISA method
The measurement of IZUMO2 albumen relative amount.Then 22 long white race boar is ranked up from high to low by reproductive efficiency, is chosen
First 11 are used as high breeding group, and latter 11 are used as low breeding group, examine breeding Growth Results and IZUMO2 sperm between two groups
Protein content whether there is significant difference.The results are shown in Table 8：The IZUMO2 sperm protein content of high breeding group is significant and pole
It is significantly higher than low breeding group, and high breeding group herd boar ginseng is with Farrowing rate, number born per litter, nest young number living and breeding
Efficiency is extremely significant to be higher than low breeding group (P<0.01).Illustrate that the relative amount of sperm protein IZUMO2 can actually reflect kind of a public affairs
The reproductive performance of pig can be used as the biomarker for screening high reproductive performance herd boar.
The reproductive performance of 8 different reproductive performance herd boar of table and the comparison of sperm protein content
Note：Same column compares, and difference capitalization Superscript letters indicate the extremely significant (P of difference<0.01), different small letter Superscript letters tables
Show significant difference (P<0.05).
(2) the screening threshold value and its application effect in herd boar breeding of IZUMO2 protein labeling
In order to establish the concrete operation method for applying the sperm protein label high reproductive performance herd boar of breeding, we are by 22
Herd boar is ranked up according to IZUMO2 protein content respectively, and compares according to different breeding levels to its creation data
Analysis, with according to breeding strength demand different in production come the high reproductive performance herd boar of breeding.
High reproductive performance herd boar breeding is carried out according to IZUMO2 protein labeling, the effect in the case where different selections are horizontal is as follows
Shown in table 9.As seen from table, good effect is all had with the high reproductive performance herd boar of IZUMO2 sperm protein content breeding.?
When 86% breeding level (boar for eliminating 14%), boar IZUMO2 relative amount of reserving seed for planting is (12.08 ± 0.83) × 10-8；
In 77% breeding level, boar IZUMO2 relative amount of reserving seed for planting is (12.64 ± 0.83) × 10-8；In 68% breeding level,
Boar IZUMO2 relative amount of reserving seed for planting is (13.19 ± 0.83) × 10-8；In 50% breeding level, boar IZUMO2 phase of reserving seed for planting
It is (14.35 ± 0.91) × 10 to content-8.When selecting and remain rate between 68% to 86%, Breeding Effect is preferable, at this time IZUMO2
Albumen relative amount range is (11.25~14.02) × 10-8.Further preferably breeding level is 68%, at this time IZUMO2 egg
White relative amount range is (12.36~14.02) × 10-8(table 9 and Fig. 4).
With the effect of IZUMO2 content breeding herd boar under the different selections of table 9 are horizontal
Note：In the case where same selection is horizontal, Superscript letters ns indicates that group difference is not significant, and * indicates that significant difference, * * indicate
Difference is extremely significant.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (8)
1. a kind of sperm protein closely related with herd boar reproductive performance marks, it is characterised in that：The sperm protein mark
It is denoted as sperm protein label IZUMO2.
2. the application of sperm protein label described in claim 1, it is characterised in that：
Application of the sperm protein label IZUMO2 in the high reproductive performance herd boar of breeding.
3. application according to claim 2, it is characterised in that：
The relative amount and herd boar reproductive performance of the sperm protein label IZUMO2 is positively correlated in significant.
4. a kind of method using the sperm protein label high reproductive performance herd boar of IZUMO2 breeding, which is characterized in that including such as
Lower step：
(1) quantitative result according to sperm protein label IZUMO2, in conjunction with various Growth Results indexs, according to different selection ratios
Example is for statistical analysis, and for each index there are when significant difference, sperm protein marks the relative amount of IZUMO2 between determining group
Value, as the reference threshold for being used for the high reproductive performance herd boar of breeding in production；
(2) sperm for acquiring breeding herd boar to be measured, extracts sperm protein sample；
(3) quantitative detection step (2) extract sperm protein sample in IZUMO2 protein labeling content, by with reference threshold
Comparison determine the height of boar reproductive performance to be measured, to select high reproductive performance herd boar, eliminate low reproductive performance
Herd boar.
5. according to the method described in claim 4, it is characterized in that：
Reference threshold described in step (1) is that breeding level is 68%~86%, at this time IZUMO2 albumen relative amount range
It is (11.25~14.02) × 10-8。
6. method according to claim 4 or 5, it is characterised in that：
Reference threshold described in step (1) is that breeding level is 68%, and IZUMO2 albumen relative amount range is at this time
(12.36~14.02) × 10-8。
7. according to the method described in claim 4, it is characterized in that：
The preparation step of sperm protein sample described in step (2) is as follows：
3mL original sperm is added in 15mL centrifuge tube in the percoll discontinuous density gradient liquid of preparation 30% and 60% on it,
400 × g is centrifuged 20min at 20 DEG C, discards supernatant, portion's Sperm pellets of only keeping on file, with DPBS centrifuge washing sperm 3 times, sperm
It counts, adjusts every pipe 5~10 × 107A sperm is added Protein Extraction Reagent according to sperm quantity, 0.5h is cracked, under the conditions of 4 DEG C
14000 × g is centrifuged 20min, takes supernatant into another centrifuge tube, and -20 DEG C of pre- cold acetones of 1.5 times of volumes, -20 DEG C of sinkings are added
It forms sediment for 24 hours, then 14000 × g is centrifuged 20min at 4 DEG C, abandons supernatant, blot raffinate, saved in -80 DEG C.
8. according to the method described in claim 4, it is characterized in that：
Quantitative detection described in step (3), which uses, utilizes BCA and ELISA method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810614803.4A CN108840919B (en) | 2018-06-14 | 2018-06-14 | Sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810614803.4A CN108840919B (en) | 2018-06-14 | 2018-06-14 | Sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108840919A true CN108840919A (en) | 2018-11-20 |
| CN108840919B CN108840919B (en) | 2020-07-03 |
Family
ID=64202370
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810614803.4A Active CN108840919B (en) | 2018-06-14 | 2018-06-14 | Sperm protein marker IZUMO2 closely related to breeding boar reproductive performance and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108840919B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110144414A (en) * | 2019-06-27 | 2019-08-20 | 广西扬翔农牧有限责任公司 | Molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods |
| CN110452997A (en) * | 2019-01-19 | 2019-11-15 | 华中农业大学 | Molecular labeling relevant to boar sperm abnormal rate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010187556A (en) * | 2009-02-16 | 2010-09-02 | Osaka Univ | Pregnancy-diagnosing method, pregnancy-diagnosing kit, polynucleotide, polypeptide, and antibody |
| CN103961718A (en) * | 2013-01-29 | 2014-08-06 | 中国人民解放军军事医学科学院生物工程研究所 | Immunological contraception vaccine and preparation method thereof |
| KR20150010881A (en) * | 2013-07-19 | 2015-01-29 | 중앙대학교 산학협력단 | Biomarkers for Predicting Fertility and Method for Predicting Fertility Using the Same |
| CN108152189A (en) * | 2017-12-06 | 2018-06-12 | 浙江大学 | A kind of sperm surface goes out the quantitative detecting method of cloud albumen 1 |
-
2018
- 2018-06-14 CN CN201810614803.4A patent/CN108840919B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010187556A (en) * | 2009-02-16 | 2010-09-02 | Osaka Univ | Pregnancy-diagnosing method, pregnancy-diagnosing kit, polynucleotide, polypeptide, and antibody |
| CN103961718A (en) * | 2013-01-29 | 2014-08-06 | 中国人民解放军军事医学科学院生物工程研究所 | Immunological contraception vaccine and preparation method thereof |
| KR20150010881A (en) * | 2013-07-19 | 2015-01-29 | 중앙대학교 산학협력단 | Biomarkers for Predicting Fertility and Method for Predicting Fertility Using the Same |
| CN108152189A (en) * | 2017-12-06 | 2018-06-12 | 浙江大学 | A kind of sperm surface goes out the quantitative detecting method of cloud albumen 1 |
Non-Patent Citations (2)
| Title |
|---|
| GRAYSON P ET AL: "Positive Selection and the Evolution of izumo Genes in Mammals", 《INT J EVOL BIOL》 * |
| 丹彤等: "绵羊Izumo2 cDNA的克隆及原核表达", 《内蒙古大学学报(自然科学版)》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110452997A (en) * | 2019-01-19 | 2019-11-15 | 华中农业大学 | Molecular labeling relevant to boar sperm abnormal rate |
| CN110452997B (en) * | 2019-01-19 | 2021-05-07 | 华中农业大学 | Molecular marker related to boar sperm teratogenesis rate |
| CN110144414A (en) * | 2019-06-27 | 2019-08-20 | 广西扬翔农牧有限责任公司 | Molecular genetic marker relevant to boar sperm abnormal rate and its application and acquisition methods |
| CN110144414B (en) * | 2019-06-27 | 2022-11-18 | 广西贵港秀博基因科技股份有限公司 | Molecular genetic marker related to boar sperm teratospermia and application and acquisition method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108840919B (en) | 2020-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Peddinti et al. | Comprehensive proteomic analysis of bovine spermatozoa of varying fertility rates and identification of biomarkers associated with fertility | |
| Dominguez et al. | Embryologic outcome and secretome profile of implanted blastocysts obtained after coculture in human endometrial epithelial cells versus the sequential system | |
| Welch et al. | Sex preselection: laboratory validation of the sperm sex ratio of flow sorted X-and Y-sperm by sort reanalysis for DNA | |
| Parrilla et al. | Boar semen proteomics and sperm preservation | |
| Pinkel et al. | Flow cytometric determination of the proportions of X-and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm | |
| Bahbahani et al. | Genome diversity and signatures of selection for production and performance traits in dromedary camels | |
| Evenson et al. | Comparison of the Halosperm® test kit with the Sperm Chromatin Structure Assay (SCSA®) infertility test in relation to patient diagnosis and prognosis | |
| Linjawi et al. | Expression of interleukin-11 receptor α and interleukin-11 protein in the endometrium of normal fertile women and women with recurrent miscarriage | |
| CN108840919A (en) | A kind of the sperm protein label IZUMO2 and its application closely related with herd boar reproductive performance | |
| Kadarmideen et al. | Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production | |
| Selvaraju et al. | Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility | |
| Barbato et al. | Purification of pregnancy-associated glycoproteins from late-pregnancy Bubalus bubalis placentas and development of a radioimmunoassay for pregnancy diagnosis in water buffalo females | |
| Myromslien et al. | Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size | |
| Kaya et al. | Sperm proteins ODF2 and PAWP as markers of fertility in breeding bulls | |
| Vogiatzi et al. | Hemizona assay and sperm penetration assay in the prediction of IVF outcome: a systematic review | |
| US7906702B2 (en) | Categorically ranking animals for feed efficiency | |
| Tsoi et al. | Telencephalic outputs from the medial entorhinal cortex are copied directly to the hippocampus | |
| CN108872588A (en) | A kind of the sperm protein label SPACA4 and its application closely related with herd boar reproductive performance | |
| US20050221322A1 (en) | Multiple closed nucleus breeding for swine production | |
| Zamfirescu et al. | mTORC1/2 signaling is downregulated by amino acid-free culture of mouse preimplantation embryos and is only partially restored by amino acid readdition | |
| Jo et al. | Effect of antibodies binding to Y chromosome-bearing sperm conjugated with magnetic nanoparticles on bull sperm characteristics | |
| CN108835031A (en) | A kind of method of the high reproductive performance herd boar of breeding | |
| Gunjača et al. | Predictive value of 8 genetic loci for serum uric acid concentration | |
| Pasandideh et al. | Identification of two novel SNPs affecting lambing traits in sheep by using a 50K SNP-Chip | |
| Lone et al. | Spermatozoa molecules in relation to bulls fertility |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230801 Address after: 518000 Fubao community, Fubao street, Futian District, Shenzhen City, Guangdong Province Patentee after: SHENZHEN EVERSUN BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 510642 No. five, 483 mountain road, Guangzhou, Guangdong, Tianhe District Patentee before: SOUTH CHINA AGRICULTURAL University |