CN111876497B - Method for identifying or assisting in identifying quality of animal semen - Google Patents

Abstract

The invention discloses a method for identifying or assisting in identifying the quality of animal semen. The method is to detect the expression quantity of miR-222 in SPEVs of an animal individual to be detected, and if the expression quantity of miR-222 in SPEVs of the animal individual to be detected is higher than the expression quantity of miR-222 in SPEVs of a control animal individual, the quality of animal semen is poor; if the expression level of the miR-222 in the SPEVs of the animal subject to be tested is not higher than the expression level of the miR-222 in the SPEVs of the control animal subject, the quality of the animal semen is good; the control animal individuals are animal individuals with good semen quality. Experiments prove that the quality of animal semen can be identified only by detecting the expression quantity of miR-222 in animal individual SPEVs. The invention has great application value for establishing a group with excellent semen quality.

Description

Method for identifying or assisting in identifying quality of animal semen

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for identifying or assisting in identifying the quality of animal semen.

Background

The RNA-seq technique, i.e., the transcriptome sequencing technique, is a technique for sequencing total RNA in cells or tissues by means of a second-generation high-throughput sequencing method. RNA-seq technology allows the detection of gene expression in cells or tissues, while important genetic variations present in the population can be detected by alignment with reference genomic sequences. At present, the RNA-seq technology is widely applied to the research of important economic traits of animals and plants.

The boar semen quality character is a quantitative character controlled by a micro-effect polygene, but the heritability is only 0.20, which belongs to the middle lower part, and the character expression is late. The semen quality is very key for improving the conception rate of sows, the litter size, the birth weight of piglets and the like. The traditional method is difficult to quickly and accurately select the semen quality, but the Marker Assisted Selection (MAS) can effectively accelerate the breeding process, select and reserve dominant groups, eliminate inferior boars and improve the semen quality characters of the boars. In recent years, with the continuous progress of the biological science technology, various omics technologies are changing day by day, and the research of transcriptomics, proteomics, metabonomics and the like is emerging in succession, which opens the post-genome era of life science research. Sperm motility is an important index for sperm quality detection.

Extracellular Vesicles (EVs) are spherical membrane vesicles actively released by cells, which are surrounded by lipid bilayers and have a diameter of 30-4000nm, and include Microvesicles (MV), apoptotic bodies (apoptotic bodies), Extracellular vesicles (exosomes), and mobilities (migraosomes). Seminal plasma contains a large number of extracellular vesicles. The prostate is a major secretory organ of the seminal plasma extracellular vesicles, and is also derived from a plurality of tissues and organs such as epididymis and other accessory gonads, so in recent years, these extracellular vesicles with complex components in seminal plasma are collectively called Seminal Plasma Extracellular Vesicles (SPEVs). Since the first discovery of SPEVs, research on its function has never been stopped. It has been found that SPEVs may be involved in the acrosome reaction to help the sperm gain energy at the correct time point, and it is believed by the scholarian that this function is achieved by inducing tyrosine phosphorylation of the sperm, and it has been found that SPEVs may protect the sperm against immune reactions in the female reproductive tract, which is important for the sperm to successfully cross the zona pellucida and bind to the ovum, and it is speculated that this function may be associated with non-coding RNA therein. SPEVs also promote sperm motility, a function which has been demonstrated by several research teams and has been demonstrated in different species, presumably by modulating the concentration of certain divalent cations such as calcium, zinc and magnesium in the sperm microenvironment to regulate the oscillation of sperm flagella. Also, studies have shown that some small non-coding RNAs in SPEVs may bind to target cells in the female reproductive tract to affect expression of the relevant gene. The study of SPEVs content is also endless, and the most extensive studies now include proteins and small non-coding RNAs (e.g., microRNAs, YRNAs, tRNAs), and much has focused on "comparative omics" to explore certain substances in SPEVs as biomarkers for certain diseases such as general infertility, obstructive and non-obstructive azoospermia, asthenospermia, prostate cancer, etc., to make the diagnosis of diseases more rapid and noninvasive. Abu-Halima and the like carry out sequencing analysis on microRNAs in SPEVs of 12 normal males and 12 oligospermia patients to identify 36 differentially expressed microRNAs in total, and a qPCR verification result shows that the expressions of miR-765 and miR-1275 in the SPEVs are obviously increased in asthenospermia patients, and the expression of miR-15a is obviously reduced. Barcel Lou et al analyzed the expression profiles of microRNAs in SPEVs among three groups of samples of patients with azoospermia and obstructive azoospermia caused by normal males and spermatogenesis disturbance, and found that the expression profiles of microRNAs are different between fertile males and infertile males and between spermatogenesis disturbance type azoospermia males and obstructive azoospermia. Murdica et al co-incubate sperm from normal males, from asthenospermia, from infertile males after vasectomy, and from normal males, and found that sperm still bound to SPEVs in vitro, consistent with Du's findings. SPEVs in normal and vasectomized males can affect sperm motility and capacitation, increase sperm motility and promote acrosome reactions, and SPEVs in asthenospermia cause sperm motility reduction, the authors speculate that the transfer of cysteine-rich secreted protein 1 from SPEVs to sperm may play a role in these phenomena. In 2013, Lidia et al explored the effect of pig SPEVs on pig capacitation, and found that it could inhibit the disappearance of the 14-kD phosphorylation signal specific to pig capacitation, and inhibit the loss of acrocholesterol and increase in fluidity during capacitation. Du et al found that pig SPEVs bind to mature sperm in vitro and promote sperm motility and maintain acrosome integrity.

There is currently no report on studies on porcine SPEVs transcriptomics, nor is there any report on how SPEVs regulate mature sperm motility. Therefore, by exploring miRNAs information in pig SPEVs, the activity of pig sperms is regulated and controlled, the miRNAs are applied to breeding boars for selective elimination, the production performance of the breeding boars can be improved, the semen utilization rate is increased, the conception rate of sows is improved, and the breeding process is accelerated.

Disclosure of Invention

The invention aims to identify or assist in identifying the quality of animal semen.

The invention firstly protects the application of a substance for detecting the expression level of miR-222, which can be at least one of a1) to a 4):

a1) identifying or assisting in identifying the quality of animal semen;

a2) preparing a product for identifying or assisting in identifying the quality of animal semen;

a3) screening or auxiliary screening of animal individuals with different semen qualities;

a4) preparing products for screening or assisting in screening of individual animals with different semen qualities.

The invention also protects application of substances and devices for detecting the expression level of miR-222, which can be at least one of a1) to a 4):

a1) identifying or assisting in identifying the quality of animal semen;

a2) preparing a product for identifying or assisting in identifying the quality of animal semen;

a3) screening or auxiliary screening of animal individuals with different semen qualities;

a4) preparing products for screening or assisting in screening of individual animals with different semen qualities;

the device can be a device A and/or a device B;

the device A can comprise a data input device 1, a data recording module 1, a data comparison module 1-1 and a conclusion output module 1-1;

the data input device 1 is used for inputting the expression quantity value of miR-222;

the data recording module 1 is used for storing the expression quantity value of miR-222;

the data comparison module 1-1 is used for comparing the expression quantity of miR-222 in the SPEVs of the animal subject to be tested with the expression quantity of miR-222 in the SPEVs of the animal subject to be controlled;

the conclusion output module 1-1 is used for displaying a conclusion, namely if the expression quantity of the miR-222 in the SPEVs of the animal subject to be tested is higher than the expression quantity of the miR-222 in the SPEVs of the control animal subject, the conclusion output module 1-1 displays that the quality of the animal semen is poor; if the expression level of the miR-222 in the SPEVs of the animal individual to be detected is not higher than the expression level of the miR-222 in the SPEVs of the control animal individual, the conclusion output module 1-1 shows that the quality of the animal semen is good;

the control animal individual can be an animal individual with excellent semen quality;

the device B can comprise data input equipment 1, a data recording module 1, a data comparison module 1-2 and a conclusion output module 1-2;

the data comparison module 1-2 is used for comparing the expression quantity of miR-222 in SPEVs of a plurality of animal individuals;

conclusion output module 1-2 is used to show the conclusion that the lower the expression level of miR-222 in SPEVs in an animal subject, the better the semen quality.

The device A can be composed of the data input device 1, the data recording module 1, the data comparison module 1-1 and the conclusion output module 1-1.

The device B can be specifically composed of the data input equipment 1, the data recording module 1, the data comparison module 1-2 and the conclusion output module 1-2.

In the present example, the control animal subject may be specifically a white pig, in the examples numbered H11, H12, H13, H14, H15 or H16, or a duroc pig, in the examples numbered H1, H2, H3 or H4.

In any of the above applications, the substance for detecting the expression level of miR-222 can be a primer pair 1 for reverse transcription (i.e. for reverse transcription of miR-222) and/or a primer pair 2 for real-time fluorescence quantitative PCR detection;

the primer pair 1 can be composed of SEQ ID NO: 1 and primer F1 shown in SEQ ID NO: 2, and a primer R1;

the primer pair 2 can be composed of SEQ ID NO: 3 and primer F3 shown in SEQ ID NO: 4, and a primer R4 shown in the specification.

The invention also provides a kit which can comprise a substance for detecting the expression quantity of miR-222; the function of the kit can be a1) or a 3):

a1) identifying or assisting in identifying the quality of animal semen;

a3) screening or auxiliary screening of animal individuals with different semen qualities.

In the kit, the substance for detecting the expression level of miR-222 can be a primer pair 1 for reverse transcription (namely reverse transcription miR-222) and/or a primer pair 2 for real-time fluorescent quantitative PCR detection;

the primer pair 1 can be composed of SEQ ID NO: 1 and primer F1 shown in SEQ ID NO: 2, and a primer R1;

the primer pair 2 can be composed of SEQ ID NO: 3 and primer F3 shown in SEQ ID NO: 4, and a primer R4 shown in the specification.

Any one of the kits can specifically consist of a substance for detecting the expression quantity of miR-222.

The invention also protects at least one of D1) -D6):

D1) the application of miR-222 as a marker in identification or auxiliary identification of animal semen quality;

D2) the application of miR-222 as a marker in the preparation of products for identifying or assisting in identifying the quality of animal semen;

D3) the application of miR-222 as a marker in screening or auxiliary screening of animal individuals with different semen qualities;

D4) the application of miR-222 as a marker in preparing products for screening or assisting in screening animal individuals with different semen qualities;

D5) the application of miR-222 as a marker in regulating and controlling animal semen quality;

D6) application of miR-222 as a marker in preparation of products for regulating animal semen quality.

The invention also protects the application of the substance for reducing the expression quantity of miR-222 in animal individual SPEVs in improving the quality of animal semen.

The invention also protects the application of the substance for improving the expression quantity of miR-222 in animal individual SPEVs in reducing the quality of animal semen.

The invention also provides a method for identifying or assisting in identifying the quality of animal semen, which comprises the following steps:

(1-1) detecting the expression quantity of miR-222 in SPEVs of an animal individual to be detected;

(1-2) judging the quality of the animal semen according to the expression quantity of miR-222;

the principle of judging the semen quality according to the expression quantity of miR-222 is as follows: the lower the expression level of miR-222, the better the semen quality.

The method can be realized by comparing the expression quantity of the miR-222 in the SPEVs of the animal to be tested with the expression quantity of the miR-222 in the SPEVs of the animal with excellent semen quality. When the animal is a pig, the animal individual with excellent semen quality can be a large white pig with the number of H11, H12, H13, H14, H15 or H16 in the examples, or a Duroc pig with the number of H1, H2, H3 or H4.

The method can be realized by comparing the expression quantity of the miR-222 in the SPEVs of the animal to be tested with the expression quantity of the miR-222 in the SPEVs of the animal with poor semen quality. When the animal is a pig, the animal subject with poor semen quality can be a large white pig with the number of L11, L12, L13, L14, L15 or L16 in the examples, or a Duroc pig with the number of L1, L2, L3 or L4.

Any of the animals described above may be any of b1) through b 4); b1) a human; b2) a pig; b3) duroc pigs; b4) big white pig.

The semen of any one of the above is excellent in quality, and can be more than 90% of the total sperm motility and more than 70% of the rapid movement.

The semen of any one of the above is of good quality, and can be greater than 90% of the total sperm motility, greater than 70% of the rapid motility, and less than 12% of the slow motility.

Any of the above-described sperm qualitatively poor may be a sperm total motility of <80% and a rapid motility of < 55%.

Any of the above-described sperm inferiorities can be <80% total sperm motility, <55% rapid motility, and >15% slow motility.

The nucleotide sequence of any one of the miR-222 is shown as SEQ ID NO: 5, respectively.

Experiments prove that in Duroc pigs or white pigs, the expression level of miR-222 in the group with high semen quality is obviously lower than that of miR-222 in the group with low semen quality. Therefore, animal semen quality can be identified by merely detecting the expression level of miR-222 in animal individual SPEVs. The invention can screen and obtain animal individuals with excellent semen quality, has higher accuracy, can be used for early screening of breeding boars, and can accurately screen even when the breeding boars are rigidly mature, thereby greatly accelerating the breeding process of the boars, selecting and reserving dominant groups, eliminating inferior boars and improving the semen quality characters of the boars. The invention has great application value for establishing the breeding boar group with excellent semen quality.

Drawings

FIG. 1 shows the expression quantity analysis of miR-222 in the semen quality high group and the semen quality low group in the transcriptome sequencing result.

FIG. 2 shows the expression level of miR-222 in Duroc SPEVs by fluorescent quantitative PCR detection.

FIG. 3 shows that the expression level of miR-222 in SPEVs of white pigs is detected by fluorescent quantitative PCR.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1, transcriptome sequencing finds that the expression of miR-222 in Duroc porcine seminal plasma extracellular vesicles with high and low semen quality has significant difference

First, test animal and group

The fresh concentrate sample used in this example was obtained from Henan Jingwang improved boar, Duroc, 24-36 months old, and was raised to sexual maturity under normal nutritional levels and feeding conditions. And (4) selecting the boars with high semen quality and the boars with low semen quality according to the semen collection records of the boar stations within half a year, and finally selecting 8 individuals. All boars were divided into two groups, high semen quality group (total vigor >90%, fast movement >70% and slow movement < 12%) and low semen quality group (total vigor <80%, fast movement <55% and slow movement > 15%). Fresh semen samples were collected by 8 boars at the same time by the semen collector.

The information on the number of 8 boars, individual number, age of month, total sperm motility (%), rapid movement (%), and slow movement (%) of fresh sperm samples is shown in table 1.

TABLE 1

Note: the serial number contains H and belongs to a group with high semen quality; the ones containing L in the numbers belong to the group of low semen quality.

Second, separation of porcine seminal plasma extracellular vesicles (i.e., porcine SPEVs)

Corresponding porcine SPEVs were isolated from 8 fresh sperm samples, respectively. The steps for isolating porcine SPEVs for each fresh sperm sample are as follows:

1. taking a fresh essence sample, balancing for 2h at 17 ℃, then centrifuging for 15min at 800g at 17 ℃, and collecting supernatant 1.

2. After step 1, the supernatant was centrifuged at 10000g for 30min at 1, 4 ℃ and collected as supernatant 2.

3. After step 2, the supernatant was centrifuged at 12000g for 60min at 2, 4 ℃ and collected as supernatant 3.

4. After completion of step 3, the supernatant was centrifuged at 120000g for 1.5h at 3, 4 ℃ and discarded, and the pellet was resuspended in DPBS buffer (Gibco, USA) to obtain a resuspension solution.

5. After completion of step 4, the resuspension was centrifuged at 120000g for 1.5h at 4 ℃ and the supernatant discarded, the pellet resuspended in DPBS buffer and then sterilized by filtration through a 0.22 μm filter to give pig SPEVs.

Pig SPEVs are stored at-80 ℃ until use.

Third, total RNA extraction and concentration determination in pig SPEVs

And extracting total RNA from the 8 porcine SPEVs separated in the step two respectively to obtain the total RNA of the porcine SPEVs. The steps for total RNA extraction for each porcine SPEVs are as follows:

1. add 200. mu.L of pig SPEVs to 700. mu.L of QIAzol lysate (QIAGEN, Germany) and vortex and mix; standing at room temperature (15-25 deg.C) for 5 min.

2. After completion of step 1, 140. mu.L of chloroform was added, covered tightly with a cover and shaken vigorously for 15s, left to stand at room temperature for 2-3min, and centrifuged at 12000g for 15min at 4 ℃.

3. After the completion of step 2, the supernatant was transferred to a new EP tube, and anhydrous ethanol of 1.5 times the volume was added thereto and mixed to obtain a mixed solution.

4. After completion of step 3, the mixture was transferred to an RNeasy Mini column (QIAGEN, germany) and placed in a 2mL collection tube. Centrifuge at 8000g for 15s at room temperature, and discard the liquid in the collection tube.

If the volume of the mixed solution obtained in the step 3 is large, the mixed solution can be transferred to an RNeasy Mini column for multiple times and collected by centrifugation.

5. After completion of step 4, 700. mu.L of Buffer RWT (QIAGEN, Germany) was added to the RNeasy Mini column, centrifuged at 8000g for 15s at room temperature, and the filtrate was discarded.

6. After completing step 5, repeating the following steps twice: 500. mu.L of Buffer RPE (QIAGEN, Germany) was added to the RNeasy Mini column, centrifuged at 8000g for 15s at room temperature, and the filtrate was discarded.

7. After completion of step 6, the RNeasy Mini column was transferred to a new 2mL collection tube and centrifuged at 12000g for 2min at room temperature.

8. After completion of step 7, the RNeasy Mini column was transferred into a new 1.5mL EP tube (QIAGEN, Germany), uncapped, and allowed to stand at room temperature for 3 min.

9. After completion of step 8, 35. mu.L of RNA free water was added to the RNeasy Mini column, covered and left to stand at room temperature for 2 min; then, centrifugation is carried out at room temperature and 12000g for 1min, and RNeasy Mini column is discarded, thus obtaining the total RNA of the pig SPEVs in EP.

Total RNA purity and concentration of 8 porcine SPEVs was measured using a Nanodrop nucleic acid Analyzer (Thermo Scientific, USA). Total RNA integrity was tested for 8 porcine SPEVs using Agilent2100(Agilent, USA).

The results indicate that the total RNA concentrations of 8 porcine SPEVs were greater than 8 ng/. mu.L, and the RIN values were 2.6. Subsequent experiments can be performed.

Construction and sequencing of four-microRNAs library

1. And (4) respectively taking the total RNA of the 8 pig SPEVs extracted in the third step, and carrying out microRNAs library establishment by Beijing Enzekangtai biology Co.

Libraries of microRNAs per pig SPEVs were prepared using the QIAseq miRNA Library Kit (QIAGEN, Germany) with simplified Library construction steps as follows: connecting a3 'end and a 5' end adaptor; ② Biotinylated Random Primers and mRNA mixed reverse transcription; ③ mRNA is captured by magnetic beads, and cDNA purification is carried out after elution; and fourthly, PCR amplification. And after library construction is finished, performing quality inspection by using an Agilent Bioanalyzer 2100, performing double-end sequencing by using Illumina Hiseq2500 after the quality inspection is qualified, and reading the sequence to obtain a microRNAs library of the pig SPEVs, wherein the sequence reading length is single-ended 10 multiplied by 150 bp.

2. Data for the microRNAs library per porcine SPEVs were analyzed as follows:

taking sequenced original sequences (Raw reads), filtering by CutAdapt software, and removing a linker sequence and low-quality reads added during library building to form Clean reads; clean reads are aligned to a Silva database, a GtRNAdb database, a Rfam database and a Repbase database by using Bowtie software, ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), small nucleolar RNA (snorNA) and repeated sequences are filtered out, unantained reads are obtained for subsequent analysis after filtering, and then the Clean reads are aligned to a reference genome sequence sscrofa11.1 (website: ftp:// ftp. ensembl. org/pub/release-97/fa/sus _ scrofa /) of a pig and a corresponding gene annotation file (sscrofa11.1) by using BWA software.

The sequencing data volume, quality control results (i.e., Q30) and alignment rate with the porcine reference genome for the 8-head boar seminal plasma extracellular vesicles are shown in table 2. The base quality value Q30 was at a level at which the base matching accuracy was 99.9%, and Q30 was at a value of Q30 or more per total base.

TABLE 2

Individual numbering	Raw_reads	Clean_reads	Q30(％)	Comparison with reference genome (%)
					H1	21616667	18065377	95.62	84.02％
H2	18250626	15496475	96.16	82.53％
					H3	19848044	16305474	95.47	84.61％
H4	17993588	13997314	95.47	79.43％
					L1	20749226	17267924	96.20	84.12％
L2	21310754	18274556	95.62	83.57％
					L3	21821253	18460052	96.17	80.65％
L4	36922802	28818583	96.03	80.06％

Screening of differential microRNA in transcriptome sequencing

1. And according to the sequencing data obtained in the fourth step, comparing the reads aligned to the reference genome with mature microRNAs sequences in a known microRNAs database miRBase by using miRDeep2 to obtain known microRNAs and predict new microRNAs. The expression quantity of the microRNAs in each sample is counted, and the expression quantity is normalized by using a TPM (Transcripts Per Million, TPM) algorithm.

The normalized formula is: TPM (Read counts × 1000000)/Mapped Reads

In the formula, read counts represent the number of Reads aligned to a certain microRNA, and Mapped Reads represents the number of Reads aligned to all microRNAs.

In addition, in consideration of the fact that low-expression microRNAs have little biological significance and high false positive, and in order to obtain more meaningful functional annotation analysis results, only microRNAs with the average expression level (TPM) higher than 10 in each sample are selected for differential expression analysis in the research.

2. Differential microRNAs between high and low groups were analyzed using edgeR software. The study sets the significance threshold for the difference to be equal to or less than 0.05.

The analytical results are shown in FIG. 1. The result shows that miR-222 is a miRNAs with extremely obvious difference in seminal plasma extracellular vesicles of porcine semen, and the expression level of miR-222 in a group with high semen quality is obviously lower than that of miR-222 in a group with low semen quality.

The nucleotide sequence of miR-222 is as follows: 5'-AGCUACAUCUGGCUACUGGGUCUC-3' (SEQ ID NO: 5).

Example 2, real-time fluorescence quantitative PCR detection of miR-222 expression level in total RNA (obtained in step three in example 1) of Duroc pig SPEVs with high and low semen quality

First, preparation of primer pairs

A primer pair 1 (consisting of a primer F1 and a primer R1) for reverse transcription and a primer pair 2 (consisting of a primer F2 and a primer R2) for real-time fluorescent quantitative PCR detection are designed and synthesized according to the nucleotide sequence of miR-222.

The nucleotide sequences of the primers are shown in Table 3.

TABLE 3

Second, obtaining cDNA of porcine SPEVs

The total RNA of the porcine SPEVs obtained in the third step of example 1 is respectively taken and reverse transcribed by using the primer pair 1 to obtain cDNA of the porcine SPEVs.

The reaction system was 15. mu.L each including 5. mu.L of total RNA of porcine SPEVs, 0.15. mu.L of 100mM dNTP, 1. mu.L of MultiScripte Reverse Transcription, 1.5. mu.L of 10 × Reverse Transcription Buffer, 1.5. mu.L of an aqueous solution of primer F1 (concentration of 10. mu.M), 1.5. mu.L of an aqueous solution of primer R1 (concentration of 10. mu.M), and 4.35. mu.L of nucleic-free H₂O。

The reaction procedure is as follows: 30min at 16 ℃; 30min at 42 ℃; 5min at 85 ℃.

Both MultiScriptbe Reverse Transcription and 10 × Reverse Transcription Buffer

Components of Kit 200Reactions Kit (ABI, USA).

Thirdly, detecting the expression quantity of miR-222 by real-time fluorescent quantitative PCR

Real-time fluorescence quantification detects the expression level of miR-222 in cDNA of 8 porcine SPEVs respectively.

The steps for real-time fluorescent quantitative detection of the expression level of miR-222 in cDNA of each pig SPEVs are as follows:

1. porcine SPEVs cDNA was taken and diluted to 300 ng/. mu.L with water to obtain a dilution of porcine SPEVs cDNA.

2. Taking a diluent of the pig SPEVs cDNA as a template, carrying out real-time fluorescence quantitative detection by adopting a primer pair 2, and then averaging according to groups to obtain the expression quantity of miR-222 in a group with high pig semen quality and a group with low pig semen quality.

The reaction system was 20. mu.L, and included 2.0. mu.L of a dilution of porcine SPEVs cDNA, 1.0. mu.L of primer F2 in water (10. mu.M), 1.0. mu.L of primer R2 in water (10. mu.M), 10.0. mu.L of SYBR PCR buffer, and 6.0. mu.L of ddH₂O。

SYBR PCR buffer is LightCycler from Roche, Switzerland

SYBR Green I Master product.

The reaction procedure is shown in Table 4.

TABLE 4

The results are shown in FIG. 2. The result shows that the expression level of miR-222 in the group with high semen quality in Duroc pigs is obviously lower than that of miR-222 in the group with low semen quality (P < 0.001).

Example 3 real-time fluorescent quantitative PCR detection of expression quantity of miR-222 in total RNA of SPEVs of large white pigs with high and low semen quality

First, preparation of primer pairs

The same procedure as in the first step of example 2.

Second, obtaining cDNA of porcine SPEVs

1. The fresh semen sample used in this example is from the south Henan Jingwang pig improvement Co., Ltd, the boar is a white pig, the month of age is 24-36 months, and the boar is raised to sexual maturity under normal nutrition level and raising conditions. And (3) selecting boars with high semen quality and boars with low semen quality according to semen collection records of boar stations within half a year, and finally selecting 12 individuals. All boars were divided into two groups, high semen quality group (total vigor >90% and fast movement > 70%) and low semen quality group (total vigor <80% and fast movement < 55%). The 12 boars were collected fresh semen samples at the same time by the semen collection staff.

The information on the 12 boars' numbers, individual numbers, age of month, total sperm motility (%), rapid movement (%), and slow movement (%) is shown in table 5.

TABLE 5

Note: the serial number contains H and belongs to a group with high semen quality; the ones containing L in the numbers belong to the group of low semen quality.

2. Isolation of porcine seminal plasma extracellular vesicles (i.e., porcine SPEVs)

Corresponding porcine SPEVs were isolated from 12 fresh sperm samples, respectively. The step of isolating porcine SPEVs for each fresh sperm sample is shown in step two of example 1.

3. Total RNA extraction and concentration determination in porcine SPEVs

Total RNA was extracted from the 12 porcine SPEVs separated in step 2, respectively, to obtain total RNA of the porcine SPEVs. The procedure for total RNA extraction for each porcine SPEVs is described in example 1, step three.

Total RNA purity and concentration of 12 porcine SPEVs was determined using a Nanodrop nucleic acid analyzer. Total RNA integrity was tested for 12 porcine SPEVs using Agilent 2100.

The results indicate that the total RNA concentrations of 12 porcine SPEVs were greater than 8 ng/. mu.L, and the RIN values were 2.6. Subsequent experiments can be performed.

4. Obtaining cDNA of porcine SPEVs

And (3) respectively taking the total RNA of the pig SPEVs obtained in the step 3, and carrying out reverse transcription by using a primer pair 1 to obtain cDNA of the pig SPEVs.

The reaction system was completely identical to the reaction system of step two in example 2.

The reaction sequence was identical to that of step two in example 2.

Thirdly, detecting the expression quantity of miR-222 by real-time fluorescent quantitative PCR

Real-time fluorescence quantification detects the expression level of miR-222 in cDNA of 12 porcine SPEVs respectively.

The steps for real-time fluorescent quantitative detection of the expression level of miR-222 in cDNA of each pig SPEVs are as follows:

1. porcine SPEVs cDNA was taken and diluted to 300 ng/. mu.L with water to obtain a dilution of porcine SPEVs cDNA.

2. Taking a diluent of the pig SPEVs cDNA as a template, carrying out real-time fluorescence quantitative detection by adopting a primer pair 2, and then averaging according to groups to obtain the expression quantity of miR-222 in a group with high pig semen quality and a group with low pig semen quality.

The reaction system was completely identical to the reaction system of step three in example 2.

The reaction sequence was identical to that of step three in example 2.

The results are shown in FIG. 3. The result shows that the expression level of miR-222 in the group with high semen quality is obviously lower than that of miR-222 in the group with low semen quality (P is less than 0.001) in the large white pig.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> university of agriculture in China

<120> a method for identifying or assisting in identifying the quality of animal semen

<160> 5

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<210> 5

<211> 24

<212> RNA

<213> Artificial sequence

<400> 5

agcuacaucu ggcuacuggg ucuc 24

Claims (6)

1. The application of the substance for detecting the miR-222 expression quantity in the seminal plasma extracellular vesicles in the preparation of products for identifying or assisting in identifying the quality of Duroc pig semen;

the semen quality is excellent or poor;

the semen has good quality, namely the total sperm motility is more than 90%, the rapid movement is more than 70%, and the slow movement is less than 12%;

the poor semen quality is that the total sperm motility is less than 80%, the rapid movement is less than 55% and the slow movement is more than 15%;

if the expression quantity of the miR-222 in the Duroc seminal plasma extracellular vesicles to be detected is higher than that of the miR-222 in the control Duroc seminal plasma extracellular vesicles, the quality of the Duroc seminal fluid to be detected is poor; if the expression level of the miR-222 in the Duroc seminal plasma extracellular vesicles to be detected is not higher than that of the miR-222 in the control Duroc seminal plasma extracellular vesicles, the quality of the Duroc seminal fluid to be detected is excellent;

the control duroc pig is a duroc pig with excellent semen quality.

2. The application of the substance for detecting the expression quantity of miR-222 in the seminal plasma extracellular vesicles in the preparation of products for identifying or assisting in identifying the semen quality of white pigs;

the semen quality is excellent or poor;

the semen has good quality, namely the total sperm motility is more than 90 percent and the rapid movement is more than 70 percent;

the poor semen quality is that the total sperm motility is less than 80% and the rapid movement is less than 55%;

if the expression quantity of the miR-222 in the seminal plasma extracellular vesicles of the large white pigs to be detected is higher than that of the miR-222 in the seminal plasma extracellular vesicles of the control large white pigs, the semen quality of the large white pigs to be detected is poor; if the expression quantity of the miR-222 in the seminal plasma extracellular vesicles of the large white pigs to be detected is not higher than that of the miR-222 in the seminal plasma extracellular vesicles of the control large white pigs, the semen quality of the large white pigs to be detected is excellent;

the control big white pig is a big white pig with excellent semen quality.

3. Use according to claim 1 or 2, characterized in that: the substances for detecting the miR-222 expression quantity in the seminal plasma extracellular vesicles are a primer pair 1 for reverse transcription and a primer pair 2 for real-time fluorescent quantitative PCR detection;

the primer pair 1 consists of SEQ ID NO: 1 and primer F1 shown in SEQ ID NO: 2, and a primer R1;

the primer pair 2 consists of SEQ ID NO: 3 and primer F3 shown in SEQ ID NO: 4, and a primer R4 shown in the specification.

4. The application of the substance and the device for detecting the expression quantity of miR-222 in the seminal plasma extracellular vesicles in the preparation of products for identifying or assisting in identifying the quality of Duroc pig semen; the semen quality is excellent or poor;

the device comprises data input equipment 1, a data recording module 1, a data comparison module 1-1 and a conclusion output module 1-1;

the data input device 1 is used for inputting the expression quantity value of miR-222;

the data recording module 1 is used for storing the expression quantity value of miR-222;

the data comparison module 1-1 is used for comparing the expression quantity of miR-222 in the Duroc pig seminal plasma extracellular vesicles to be detected with the expression quantity of miR-222 in control Duroc pig seminal plasma extracellular vesicles;

the conclusion output module 1-1 is used for displaying a conclusion, namely if the expression quantity of the miR-222 in the extracellular vesicles of the Duroc pig seminal plasma to be detected is higher than the expression quantity of the miR-222 in the extracellular vesicles of the control Duroc pig seminal plasma, the conclusion output module 1-1 displays that the quality of the Duroc pig seminal fluid is poor; if the expression level of miR-222 in the Duroc seminal plasma extracellular vesicles to be detected is not higher than that of miR-222 in the control Duroc seminal plasma extracellular vesicles, the conclusion output module 1-1 shows that the quality of the Duroc seminal fluid is excellent;

the control duroc pig is a duroc pig with excellent semen quality;

the semen has good quality, namely the total sperm motility is more than 90%, the rapid movement is more than 70%, and the slow movement is less than 12%;

the poor semen quality is sperm total motility <80%, rapid motility <55% and slow motility > 15%.

5. The application of the substance and the device for detecting the expression quantity of miR-222 in the seminal plasma extracellular vesicles in the preparation of products for identifying or assisting in identifying the quality of the semen of the white boar; the semen quality is excellent or poor;

the device comprises data input equipment 1, a data recording module 1, a data comparison module 1-1 and a conclusion output module 1-1;

the data input device 1 is used for inputting the expression quantity value of miR-222;

the data recording module 1 is used for storing the expression quantity value of miR-222;

the data comparison module 1-1 is used for comparing the expression quantity of miR-222 in the seminal plasma extracellular vesicles of the large white pigs to be detected with the expression quantity of miR-222 in the seminal plasma extracellular vesicles of the control large white pigs;

the conclusion output module 1-1 is used for displaying a conclusion, namely if the expression quantity of the miR-222 in the seminal plasma extracellular vesicles of the large white pigs to be detected is higher than the expression quantity of the miR-222 in the seminal plasma extracellular vesicles of the control large white pigs, the conclusion output module 1-1 displays that the semen quality of the large white pigs is poor; if the expression quantity of the miR-222 in the seminal plasma extracellular vesicles of the white pigs to be detected is not higher than that of the miR-222 in the seminal plasma extracellular vesicles of the control white pigs, the conclusion output module 1-1 shows that the quality of the seminal fluid of the white pigs is excellent;

the control big white pig is a big white pig with excellent semen quality;

the semen has good quality, namely the total sperm motility is more than 90 percent and the rapid movement is more than 70 percent;

the poor semen quality is sperm total motility <80% and rapid movement < 55%.

6. Use according to claim 4 or 5, characterized in that: the substances for detecting the miR-222 expression quantity are a primer pair 1 for reverse transcription and a primer pair 2 for real-time fluorescent quantitative PCR detection;

the primer pair 1 consists of SEQ ID NO: 1 and primer F1 shown in SEQ ID NO: 2, and a primer R1;

the primer pair 2 consists of SEQ ID NO: 3 and primer F3 shown in SEQ ID NO: 4, and a primer R4 shown in the specification.

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