Biomarker, preparation method and application thereof, and method for assisting breeder cock semen quality identification

Abstract

The invention relates to the technical field of cock breeding, in particular to a biomarker, a preparation method and application thereof and a method for assisting in breeding cock semen quality identification. The biomarker comprises the following components: phosphatidylcholine, lysophosphatidylcholine, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidylserine and lysophosphatidylserine. According to the invention, key markers can be rapidly detected from the semen so as to determine breeding suitable for liquid-state preservation of the semen and high-activity individuals. The artificial insemination interval time can be greatly prolonged, and long-time high-sperm active breeding cock individuals can be rapidly bred. The breeder cock breeding amount can be reduced, grains are saved, and breeding benefits are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of rooster breeding, and in particular to a biomarker, a preparation method and application thereof, and a method for assisting in the quality identification of rooster semen. Background Art

[0002] With the widespread application of artificial insemination technology in the scale and industrial development of poultry, whether in commercial chicken breeding or in the process of local chicken seed preservation, the semen quality of breeding roosters is a key factor affecting chicken artificial insemination and fertility, and the in vitro survival time of sperm is the bottleneck restricting the extension of the insemination interval.

[0003] Proper dilution and cryopreservation of rooster semen are effective ways to prolong sperm survival in vitro. Differences in sperm motility after dilution and storage are a key determinant of sperm survival in vitro. Even when the semen diluent and cryopreservation procedures are identical, significant individual differences in sperm motility after dilution and storage can significantly impact sperm survival in vitro. The effectiveness of semen cryopreservation is the result of the interaction between sperm and seminal plasma under low-temperature conditions. Seminal plasma has a decisive influence on sperm function. It contains a rich supply of non-coding RNA, proteins, metabolites, and chemical molecules, providing sperm with nutrition, energy, and a safe environment, making it a crucial factor in sperm survival in vitro.

[0004] Lipids are major components of cell membranes and play a crucial role in maintaining the structural integrity of cells. One of the essential conditions for in vitro storage of semen is low temperature, generally 2-5°C. Lipids are known to significantly influence sperm structure and function both in vivo and in vitro. Lipid changes specifically affect the phospholipid and cholesterol content of sperm membranes, impacting membrane fluidity, stability, and fusion capacity. In birds, lipids are thought to play a crucial role in in vivo storage in the female utero-vaginal gland.

[0005] Traditional semen quality analysis cannot fully diagnose the cause of changes in sperm motility. Therefore, there is an urgent need for a biomarker method to evaluate the semen quality of breeding roosters. This method can serve as an auxiliary method for identifying whether the semen of breeding roosters can be stored at 4°C for 5 days, so as to better identify or predict whether the semen of breeding roosters is suitable for liquid storage, thereby screening high-quality breeding rooster semen suitable for storage for production, which can extend the interval between artificial inseminations and has important reference value for the selection and breeding of breeding roosters. Summary of the Invention

[0006] The purpose of the present invention is to provide a biomarker, a preparation method and application thereof, and a method for assisting in the quality identification of rooster semen, so as to solve the problem that the existing rooster semen evaluation indicators can only be used for preliminary screening, but cannot quickly and accurately evaluate the motility of the same sperm and whether individual semen can be stored in liquid form.

[0007] Therefore, the present invention provides a biomarker comprising: phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, and lysophosphatidylserine.

[0008] In some embodiments of the present invention, the phosphatidylcholine includes PC (22:4 / 0:0), PC (P-18:1 (9Z) / 18:3), the hemolytic phosphatidylcholine includes LPC (18:1 (11Z) / 0:0), LPC (18:1-SN1), the phosphatidylethanolamine includes PE (18:1 (9Z) / 0:0), PE (20:4 / 0:0), PE (18:2 / 0:0), PE (18:1 / 0:0), the hemolytic phosphatidylethanolamine includes LPE (18:1), LPE (20:4), LPE (18:0), LPE (18:0 / 0:0), the phosphatidylserine includes PS (18:1 (9Z) / 0:0), and the hemolytic phosphatidylserine includes LPS (18:0).

[0009] The present invention also provides a method for preparing the above-mentioned biomarker, comprising the following steps:

[0010] The semen of roosters was separated to obtain seminal plasma, which was then subjected to metabolomics analysis to obtain differential metabolites.

[0011] In some embodiments of the present invention, the separation speed is 2500-3500 rpm, and the separation time is 3-6 min.

[0012] The present invention also provides the use of the above-mentioned biomarkers in assisting the identification of semen quality of roosters.

[0013] The present invention also provides a method for assisting in identifying the quality of rooster semen, comprising the following steps:

[0014] Separate the semen of the roosters to obtain seminal plasma;

[0015] Metabolomics technology was used to detect the levels of the following biomarkers in seminal plasma:

[0016] Phosphatidylcholine: PC(22:4 / 0:0), PC(P-18:1(9Z) / 18:3), Lysophosphatidylcholine: LPC(18:1(11Z) / 0:0), LPC(18:1-SN1), Phosphatidylethanolamine: PE(18:1(9Z) / 0:0), PE(20:4 / 0:0), PE(18:2 / 0:0), PE(18:1 / 0:0), Lysophosphatidylethanolamine: LPE(18:1), LPE(20:4), LPE(18:0), LPE(18:0 / 0:0), Phosphatidylserine: PS(18:1(9Z) / 0:0), Lysophosphatidylserine: LPS(18:0);

[0017] Multidimensional statistical analysis determined the metabolite contribution to the model grouping (VIP), and unidimensional statistical analysis determined the P value and absolute value of log2FC. If VIP > 1 in multidimensional statistical analysis and P < 0.05 in unidimensional statistical analysis, and |log2FC| ≥ 0, the semen of this rooster is of high quality and suitable for storage at 4°C for 5 days.

[0018] The present invention has the following beneficial effects

[0019] The present invention provides biomarkers, including phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, and lysophosphatidylserine. By identifying differential metabolic markers in rooster semen, it is possible to quickly determine whether a rooster's semen is suitable for storage at 4°C for 5 days, and subsequently determine whether such roosters should be selected as high-vitality breeding individuals for extended artificial insemination intervals.

[0020] This method can rapidly detect key markers in seminal plasma to determine suitable semen for liquid storage and select high-motility individuals. It can significantly extend the interval between artificial inseminations and rapidly select individual roosters with long-term high sperm motility. This can reduce the number of roosters kept, conserve food, and significantly improve breeding efficiency.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the result of the same high-motility semen after 5 days of storage at 4°C;

[0023] Figure 2 This is a graph showing the temporal trend of semen fertilization rates in the HMG and LMG groups;

[0024] Figure 3 It is the statistical graph of fertilization rate of semen of HMG group and LMG group;

[0025] Figure 4is the KEGG enrichment histogram of metabolites in the seminal plasma of the HMG group and the LMG group;

[0026] Figure 5 This is the KEGG enrichment bubble diagram of metabolites in the seminal plasma of the HMG group and the LMG group;

[0027] Figure 6 The correlation heat map of the TOP30 differential metabolites in the seminal plasma of the HMG group and the LMG group was drawn;

[0028] Figure 7 is a graph of the differential marker metabolite contents in the seminal plasma of the HMG group and the LMG group;

[0029] in, Figure 7 A in the figure is a comparison chart of seminal plasma LCAT content. Figure 7 B in the figure is a comparison chart of PC content in seminal plasma. Figure 7 C in the figure is a comparison chart of seminal plasma LPC content. Figure 7 D in the figure is a comparison chart of seminal plasma FC content. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the accompanying drawings and examples. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] The experimental animals were Yufen No. 1 roosters (147 in number, 40 weeks old, weighing 2.39±0.27, purchased from the Poultry Breeding Resources Farm of Henan Agricultural University).

[0033] The semen of the breeding roosters was collected by dorsal and abdominal massage. Each rooster was collected three times with an interval of one day between each collection. The mixed samples were used as semen samples.

[0034] The semen samples were diluted 1:1 and stored at 4°C for 5 days. Figure 1 According to the results of the motility test, sperm with a motility of ≥0.6 were identified as the high motility sperm group (HMG), and sperm with a motility of ≤0.1 were identified as the low motility sperm group (LMG).

[0035] The semen of the HMG group and LMG group were used for artificial insemination experiments. The trend of fertilization rate over time is as follows: Figure 2 The fertilization rate statistics are shown in Figure 3 shown.

[0036] from Figure 2 It can be seen that after insemination, the fertilization rates of semen in the HMG group and the LMG group maintained a high fertilization rate and no difference in the first 9 days, but there was a significant difference on the 10th day, that is, the fertilization rate of HMG sperm was significantly higher than that of LMG sperm, and the fertilization rate was higher than that of LMG sperm every day thereafter until the 20th day of the entire cycle. Figure 3 The following is a statistical chart of the fertilization rate for each day within 20 days after artificial insemination. It can be seen that the insemination rate of semen from the HMG group was significantly different from that of the LMG group on day 10. This shows that HMG semen is not only suitable for storage at 4°C for 5 days, but also suitable for selecting these breeding roosters as high-vitality breeding individuals for extending the interval between artificial inseminations.

[0037] Seminal plasma metabolite detection

[0038] The above-mentioned semen of the HMG group and the LMG group were taken and centrifuged at 3000 rpm for 5 minutes at a temperature of 4°C to obtain the corresponding seminal plasma of the HMG group and the LMG group. The above-mentioned seminal plasma was subjected to the content detection of metabolites and key proteins. The double-antibody one-step sandwich enzyme-linked adsorption assay (ELISA) was used. The operation was carried out according to the instructions and the absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader to calculate the sample concentration. The Elisa kit (Jiangsu Enzyme Immunity Biological Company, China) was used to determine the content of LCAT, PC, LPC, and FC in the seminal plasma and the quantitative analysis of differential metabolites. Among them, the metabolite KEGG enrichment bar graphs of the seminal plasma of the HMG group and the LMG group are shown in the figure. Figure 4 As shown in the KEGG enrichment bubble diagram of metabolites in the seminal plasma of the HMG group and the LMG group, Figure 5 As shown. The top 20 pathways with the smallest P value are displayed in a bubble chart. RichFactor represents the number of differential metabolites in the pathway / the total number of metabolites in the pathway. The larger the RichFactor value, the greater the enrichment degree of the pathway. In the scatter plot, the size of the dot represents the number of differential metabolites in the pathway, and the color of the dot represents the p value of the enrichment analysis, that is, the significance of the enrichment. Figure 4 and Figure 5 It can be seen that the metabolites in the glycerophospholipid metabolism pathway of ko00564 (Glycerophospholipid metabolism) are relatively high. The KEGG enrichment analysis bubble diagram shows that it is significantly enriched in the glycerophospholipid metabolism pathway. The protein significantly enriched in the glycerophospholipid pathway is LCAT (lecithin cholesterol acyltransferase), and the significantly enriched metabolites are PC (phosphatidylcholine) and LPC (lysophosphatidylcholine).

[0039] Cluster analysis of the TOP30 differential metabolites in the seminal plasma of the HMG group and the LMG group was performed, and a correlation heat map of the significantly differential metabolites was drawn as shown in the figure. Figure 6 As shown: Select the TOP30 metabolites with significant differences in P values ​​to draw a correlation heat map, where the redder the color, the stronger the positive correlation between metabolites, and the bluer the color, the stronger the negative correlation between metabolites. The significance of the correlation between metabolites will be calculated. The smaller the P value, the more significant the correlation. Figure 6 It can be seen that metabolites such as LPC are more clustered in LMG.

[0040] The LCAT content, PC content, LPC content and free cholesterol (FC) content in the seminal plasma of the HMG group and the LMG group were detected. Figure 7 As shown. Figure 7 As can be seen, LCAT content was significantly upregulated in LMG, LPC content was significantly higher in LMG than in HMG, and seminal plasma PC content was significantly higher in HMG than in LMG. This demonstrates that the biomarkers and method for identifying semen quality of roosters provided by the present invention can rapidly identify semen suitable for 5-day storage at 4°C, and can also screen for high-quality roosters that can extend the interval between artificial inseminations.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A biomarker, characterized in that include: Phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, lysophosphatidylserine.

2. The biomarker according to claim 1, characterized in that The phosphatidylcholine includes PC (22:4 / 0:0), PC (P-18:1 (9Z) / 18:3), the lysophosphatidylcholine includes LPC (18:1 (11Z) / 0:0), LPC (18:1-SN1), the phosphatidylethanolamine includes PE (18:1 (9Z) / 0:0), PE (20:4 / 0:0), PE (18:2 / 0:0), PE (18:1 / 0:0), the lysophosphatidylethanolamine includes LPE (18:1), LPE (20:4), LPE (18:0), LPE (18:0 / 0:0), the phosphatidylserine includes PS (18:1 (9Z) / 0:0), and the lysophosphatidylserine includes LPS (18:0).

3. A method for preparing a biomarker according to claim 1 or 2, characterized in that: The following steps are involved: The semen of roosters was separated to obtain seminal plasma, which was then subjected to metabolomics analysis to obtain differential metabolites.

4. The method for assisting in the identification of biomarkers of rooster semen quality according to claim 3, characterized in that: The separation speed is 2500-3500 rpm, and the separation time is 3-6 minutes.

5. Use of the biomarker according to claim 1 or 2 in assisting in the identification of semen quality of roosters.

6. A method for assisting in the identification of semen quality of roosters, characterized in that: The following steps are involved: Separate the semen of the roosters to obtain seminal plasma; Metabolomics technology was used to detect the levels of the following biomarkers in seminal plasma: Phosphatidylcholine: PC (22:4 / 0:0), PC (P-18:1 (9Z) / 18:3), Lysophosphatidylcholine: LPC (18:1 (11Z) / 0:0), LPC (18:1-SN1), Phosphatidylethanolamine: PE(18:1(9Z) / 0:0), PE(20:4 / 0:0), PE(18:2 / 0:0), PE(18:1 / 0:0), lysophosphatidylethanolamine: LPE(18:1), LPE(20:4), LPE(18:0), LPE(18:0 / 0:0), phosphatidylserine: PS(18:1(9Z) / 0:0), lysophosphatidylserine: LPS(18:0); The contribution value VIP of its metabolites to the model grouping was obtained through multidimensional statistical analysis, and the P value and absolute value of log2FC were obtained through unidimensional statistical analysis.

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