Method for improving application efficiency of frozen semen of sheep

Abstract

The invention discloses a method for improving the application efficiency of frozen semen of sheep, and relates to the field of animal reproduction and breeding science, and the method comprises the following steps: collecting semen, freezing half of the semen, then respectively carrying out exosome extraction and identification experiments, then carrying out pandorah sequencing on small non-coding RNA (Ribonucleic Acid), and finally obtaining the application efficiency of the frozen semen of the sheep. The small non-coding RNA in fresh semen and frozen semen is qualitatively and quantitatively analyzed, differential expression microRNAs are screened out, corresponding target genes and signal channels are analyzed, and the small non-coding RNA and the signal channels related to semen cryopreservation are screened out. Finally, the differentially expressed small non-coding RNAs are added into frozen semen in a manner of in-vitro chemical synthesis and the like, or are added in an auxiliary manner during in-vitro artificial insemination, or are delivered through lipidosome, so that the phenotype of an embryo after insemination is influenced, the conception rate of in-vitro artificial insemination is increased, the problem in practical production is favorably solved, and the economic benefit is increased. And economic values are created for enterprises.

Description

Technical Field

[0001] The present invention relates to the field of animal breeding, and particularly to a method for improving the application efficiency of frozen sheep semen. Background Art

[0002] Non-coding RNA (ncRNA) refers to a class of small RNAs that are not translated into proteins, with a length generally of 20-25 nucleotides (nt). With the development of biology and the mutual penetration of multiple disciplines, people's understanding of ncRNA has deepened day by day. In the past decade or so, a large number of new ncRNAs have been discovered. Although they do not have the function of encoding proteins, they participate in the process of protein translation and are key molecules for RNA to achieve its functions.

[0003] MicroRNA is a class of non-coding single-stranded RNA molecules encoded by endogenous genes, with a length of about 22 nucleotides, and they participate in the regulation of post-transcriptional gene expression in animals and plants.

[0004] The development of artificial insemination technology has promoted the development of the breeding industry. Especially the application of the preservation technology of frozen semen has made the artificial insemination technology more perfect. However, after semen cryopreservation, problems such as inevitable decline in motility, morphological changes, and acrosome shedding will occur, and there are also some changes that cannot be observed by the naked eye / microscope. These changes have led to a low conception rate of artificial insemination. Summary of the Invention

[0005] The present invention aims to provide a method for improving the application efficiency of frozen sheep semen to solve the problem of low conception rate of artificial insemination after semen freezing.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for improving the application efficiency of frozen sheep semen, comprising the following steps:

[0008] S1. Collect sheep semen, and subject half of it to programmed freezing;

[0009] S2. Respectively perform exosome extraction and identification experiments on fresh semen and frozen semen;

[0010] S3. Respectively perform Pandora sequencing of small non-coding RNAs on the exosomes extracted from fresh semen and frozen semen, and perform qualitative and quantitative analysis of small non-coding RNAs;

[0011] S4. Screen out differentially expressed small non-coding RNAs, perform corresponding target gene and signaling pathway analysis, and screen out small non-coding RNAs and signaling pathways related to semen cryopreservation;

[0012] S5. Add the small non-coding RNAs related to semen cryopreservation screened out by in vitro synthesis into cryopreserved semen to improve the fertilization and conception rate.

[0013] Furthermore, in step S5, assist microinjection of small non-coding RNAs related to semen cryopreservation during in vitro artificial insemination to improve the fertilization and conception rate.

[0014] Furthermore, in step S5, deliver small non-coding RNAs related to semen cryopreservation through liposomes to improve the fertilization and conception rate.

[0015] Principle and beneficial effects of this technical solution:

[0016] 1. The existing conception rate of artificial insemination with cryopreserved semen is low. After semen cryopreservation, in addition to visible changes in sperm, such as decreased motility, morphological changes, acrosome shedding, etc., some changes that cannot be observed by the naked eye / microscope are also related to the differences in sperm before and after cryopreservation. The small non-coding RNAs screened out by the present invention explain these differences in sperm before and after cryopreservation that cannot be explained by the naked eye / microscope.

[0017] 2. Since the small non-coding RNAs in exosomes are convenient for in vitro synthesis, after further chemical modification to improve their stability, they can be easily added, or assisted injection can be carried out during in vitro artificial insemination, or delivered through liposomes, so as to improve the conception rate and even affect the phenotype of embryos after fertilization, etc., which helps to solve the problems in actual production and create economic value for enterprises. Description of the Drawings

[0018] Figure 1 It is a schematic flow chart of a method for improving the application efficiency of frozen sheep semen;

[0019] Figure 2 It is a schematic diagram of the results of exosome isolation and identification;

[0020] Figure 3 It is a schematic diagram of the signal pathway enrichment analysis of small non-coding RNA target genes. Detailed Embodiments

[0021] The present invention will be further described in detail below with reference to the drawings and embodiments:

[0022] As Figure 1 shown, the present invention makes a new contribution in the direction of improving the conception rate of artificial insemination in sheep, and specifically includes the following steps:

[0023] S1. Collect sheep semen, and subject half of it to programmed cryopreservation treatment;

[0024] In this example, the semen of the crossbred F1 generation of East Friesian and Hu sheep was extracted. The three sheep used were in the same feeding environment and were all 10 months old. To avoid batch differences between ejaculations, semen from the same sheep was collected three times and then mixed. Half of the semen was frozen.

[0025] S2. Exosome extraction and identification experiments were respectively carried out on the extracted fresh semen and frozen semen;

[0026] After selecting the samples, half of the semen was subjected to conventional freezing treatment. Then, exosomes were separated and identified from the fresh semen and frozen semen respectively. Transmission electron microscopy was used for morphological analysis of semen exosomes, and western blot was used to detect the marker proteins of exosomes, such as Figure 2 As shown, the morphology and size of exosomes were good. The western blot results showed that the marker proteins CD63 and CD81 of exosomes were both positive, and Calnexin was negative.

[0027] S3. Pandora sequencing of small non-coding RNAs was respectively carried out on the exosomes extracted from fresh semen and frozen semen for qualitative and quantitative analysis of small non-coding RNAs;

[0028] S4. Differentially expressed microRNAs were screened and corresponding target genes and signaling pathways were analyzed to screen out small non-coding RNAs and signaling pathways related to semen cryopreservation;

[0029] Table 1 Differentially expressed small non-coding RNAs (down-regulated)

[0030]

[0031] Table 2 Differentially expressed small non-coding RNAs (up-regulated)

[0032]

[0033] As shown in Tables 1 - 2, the differentially expressed microRNAs in fresh semen and frozen semen were analyzed, including up-regulated microRNAs and down-regulated microRNAs. The results showed that the expression levels of oar-miR-novel-NC_056056.1_8813 and oar-miR-novel-NC_056065.1_26359 decreased significantly after semen cryopreservation. On the contrary, the expression levels of oar-miR-novel-NC_056055.1_5214 and oar-miR-novel-NC_056064.1_23938 increased significantly in exosomes after semen cryopreservation. Subsequently, the pathways enriched by the target genes of these differentially expressed microRNAs were analyzed, such as Figure 3As shown in the figure, signal pathway analysis was performed on the screened differential microRNAs, and it was found that carbohydrate metabolism, insulin, and glucagon were significantly enriched, indicating that semen cryopreservation affects the small non-coding RNA profile of exosomes, and the small non-coding RNAs related to cryopreservation are mainly related to glucose metabolism, etc., suggesting the importance of maintaining sperm energy supply during the cryopreservation process.

[0034] S5. The small non-coding RNAs screened out and related to semen cryopreservation were added to frozen semen by in vitro synthesis to improve the fertilization rate.

[0035] In this example, by screening the differentially expressed small non-coding RNAs in the exosomes of fresh semen and frozen semen and analyzing the signal pathways related to their target genes, four candidate microRNAs significantly related to freezing were screened out. The signal pathway showed that the insulin-related signal pathway was significantly affected, indicating that the small non-coding RNAs affected by the freezing process may be related to glucose metabolism / insulin, suggesting that sperm energy metabolism / vitality is affected before and after semen freezing.

[0036] The existing artificial insemination pregnancy rate of frozen sheep semen is low, and repeated artificial insemination, etc. increase the labor cost and economic input of the sheep farm / enterprise. After semen cryopreservation, in addition to the visible changes in sperm, such as decreased motility, changes in sperm membrane structure, acrosome shedding, etc., some changes that cannot be observed by the naked eye / microscope are also related to the differences in sperm before and after cryopreservation. The screened small non-coding RNAs explain these differences in sperm before and after cryopreservation that cannot be explained by the naked eye / microscope.

[0037] In addition, since the small non-coding RNAs in exosomes are convenient for in vitro synthesis, after further chemical modification to improve their stability, they can be delivered by lipid carriers, thus playing a role in improving the conception rate and even affecting the phenotype of embryos after fertilization, helping to solve practical production problems and creating economic value for enterprises.

[0038] The above are only embodiments of the present invention. Specific technical solutions and / or common knowledge such as characteristics well known in the art are not described in detail here. It should be noted that for those skilled in the art, without departing from the technical solution of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent. The protection scope required by this application should be based on the content of its claims, and the specific implementation manners described in the specification can be used to explain the content of the claims.

Claims

1. A method for improving the application efficiency of frozen semen of sheep, characterized in that, It includes the following steps: S1. Collect sheep semen, and subject half of it to programmed freezing; S2. Conduct exosome extraction and identification experiments on fresh semen and frozen semen respectively; S3. Conduct Pandora sequencing of small non-coding RNAs on the exosomes extracted from fresh semen and frozen semen respectively, and conduct qualitative and quantitative analysis of small non-coding RNAs; S4. Screen out differentially expressed small non-coding RNAs, conduct corresponding target gene and signaling pathway analysis, and screen out small non-coding RNAs and signaling pathways related to semen cryopreservation; S5. Add the screened small non-coding RNAs related to semen cryopreservation into the frozen semen by in vitro synthesis to improve the fertilization and conception rate.

2. The method for improving the application efficiency of frozen semen of sheep according to claim 1, characterized in that, In step S5, assist in injecting small non-coding RNAs related to semen cryopreservation during in vitro artificial insemination to improve the fertilization and conception rate.

3. A method for improving the application efficiency of frozen sheep semen according to claim 1, characterized in that, In step S5, deliver small non-coding RNAs related to semen cryopreservation through liposomes to improve the fertilization and conception rate.

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