Five-time concentrated diluent for sheep semen as well as use method and application of five-time concentrated diluent

Abstract

The invention discloses a sheep semen five-time concentrated diluent as well as a use method and application thereof, and belongs to the technical field of semen diluents. The concentrated diluent is prepared from the following components: soybean lecithin, fructose, Tris, bovine serum albumin, adenosine triphosphate, reduced glutathione, citric acid, vitamin C, trehalose, mycillin and water. The sheep semen five-time concentrated diluent contains soybean lecithin and other components, can significantly improve the sperm motility performance, sperm plasma membrane integrity, sperm acrosome integrity and artificial insemination conception rate of the sheep semen after low-temperature preservation, and can improve the preservation effect of the sheep semen. The technical support is provided for improving the artificial hybridization success rate and the breeding efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of semen diluents, and in particular to a 5-fold concentrated diluent of sheep semen and a use method and application thereof. Background Art

[0002] Artificial insemination technology can maximize the breeding value of high-quality rams and overcome time and regional differences for timely breeding. At the same time, semen diluents are of great significance in expanding semen capacity, low-temperature storage of semen, and improving reproductive efficiency. At present, large-scale farms mainly use artificial insemination, and sheep semen is mainly stored at low temperatures, but it may also be stored at room temperature, because the low-temperature storage effect of existing diluent formulas is unstable and difficult to promote and apply in production.

[0003] In the field of semen diluent preparation technology, diluents prepared by adding ingredients such as egg yolk have been used in semen of various animals, but egg yolk diluents cannot be sterilized at high temperature or used repeatedly by freezing and thawing, and there are risks such as biosafety. However, there are few reports on the use of non-animal-derived substances to prepare diluents. Studies have shown that excessive reactive oxygen will accumulate in semen during in vitro storage, and excessive reactive oxygen will cause lipid peroxidation and destroy the plasma membrane structure, thereby seriously affecting sperm quality and conception rate. In addition, the preparation and packaging of the diluent are carried out in the laboratory, and the volume and weight of the product will limit the convenience of the transportation process, resulting in an increase in the cost of semen storage and difficulty in promotion. Therefore, in order to improve the low-temperature storage effect of sheep semen, further improvements need to be made in the preparation technology of semen diluents. Summary of the invention

[0004] The purpose of the present invention is to provide a 5-fold concentrated diluent of sheep semen and a method and application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention prepares a 5-fold concentrated diluent of sheep semen, which comprises ingredients such as soybean lecithin, and can significantly improve the sperm motility, sperm plasma membrane integrity, sperm acrosome integrity and artificial conception rate of sheep semen after low-temperature storage, and provides technical support for improving the preservation effect of sheep semen, and improving the success rate of artificial breeding and reproductive efficiency.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides a 5-fold concentrated diluent of sheep semen. Each 35.2 mL of the concentrated diluent comprises the following components by weight: 0.7-0.8 g of soybean lecithin, 1.5-3 g of fructose, 4.8-5.0 g of Tris, 0.16-0.3 g of bovine serum albumin, 60-80 mg of adenosine triphosphate, 60-100 mg of reduced glutathione, 2.5-2.7 g of citric acid, 0.5-0.6 g of vitamin C, 0.5-0.55 g of trehalose, 0.7-0.8 g of penicillin-streptomycin, and the rest is water.

[0007] Optionally, each 35.2 mL of diluent includes the following components by weight: 0.75 g soy lecithin, 2 g fructose, 4.84 g Tris, 0.16 g bovine serum albumin, 80 mg adenosine triphosphate, 100 mg reduced glutathione, 2.68 g citric acid, 0.5 g vitamin C, 0.55 g trehalose, 0.74 g penicillin-streptomycin, and the rest is water.

[0008] Optionally, in each 35.2 mL of concentrated diluent, the potency of penicillin-streptomycin is 550,000-650,000 IU.

[0009] The present invention also provides a method for using the 5-fold concentrated diluent of sheep semen, which comprises fully mixing the 5-fold concentrated diluent of sheep semen with water, and then incubating the mixture at 37°C before use.

[0010] Optionally, the volume ratio of the 5-fold concentrated sheep semen diluent to the water is 1:4; and the incubation time is 2-4 min.

[0011] The present invention also provides the use of the 5-fold concentrated diluent of sheep semen in improving the low-temperature storage quality of sheep semen, wherein the improvement of the low-temperature storage quality of sheep semen includes improving sperm motility, improving sperm plasma membrane integrity, improving sperm acrosome integrity and improving artificial insemination conception rate.

[0012] The present invention also provides a method for cryopreserving sheep semen at low temperature, comprising the steps of diluting the sheep semen with the 5-fold concentrated diluent of the sheep semen and then cryopreserving the semen.

[0013] Furthermore, the 5-fold concentrated sheep semen diluent is diluted with water in advance before use; optionally, the volume ratio of the 5-fold concentrated sheep semen diluent to water is 1:4.

[0014] Furthermore, when diluting the sheep semen, an isothermal dilution method is adopted.

[0015] Optionally, the sheep semen is diluted 4-9 times.

[0016] The present invention discloses the following technical effects:

[0017] The present invention uses non-animal-derived soybean lecithin to prepare a 5-fold concentrated diluent of sheep semen. The preparation and application method of the 5-fold concentrated diluent of sheep semen is shown in Figure 1 The flow chart shown. The present invention first prepares a concentrated solution, which contains all the raw materials of the semen diluent and can be stored for future use. In subsequent use, pure water can be directly added in proportion. The operation is convenient and quick, and the product preparation efficiency can be effectively improved, and the product shelf life can be increased. At the same time, the prepared materials are sterilized in a water bath, which can effectively improve the hygiene of the finished product. The overall method has a good practical application effect. Compared with conventional preparation methods, this method improves the sustainability of material resources and biosafety, and each component material can be fully integrated to improve product quality.

[0018] The 5-fold concentrated diluent of sheep semen of the present invention can significantly improve the sperm motility, sperm plasma membrane integrity, sperm acrosome integrity and artificial conception rate of sheep semen after low-temperature storage, and provides technical support for improving the sheep semen storage efficiency, and improving the artificial breeding success rate and reproductive efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The present invention is a flow chart of the preparation and application method of the 5-fold concentrated diluent of sheep semen;

[0021] Figure 2 This is the test result of sperm plasma membrane integrity after 5 days of cryopreservation;

[0022] Figure 3 This is the test result of sperm acrosome integrity after 5 days of cryopreservation. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The experiments in the following embodiments of the present invention were all completed in the Key Laboratory of Animal Husbandry Science and Technology Corps and the Animal Experiment Station of Tarim University.

[0029] The reagents and consumables used in the following embodiments of the present invention include:

[0030] Soy lecithin, fructose, Tris, bovine serum albumin, adenosine triphosphate, reduced glutathione, citric acid, vitamin C, trehalose, penicillin-streptomycin, PBS, egg yolk, 4% paraformaldehyde, Hoechst, PI, FITC-PNA, antifade agent, purified water, electronic balance, computer-assisted semen analyzer (CASA), magnetic stirrer, electronic constant temperature incubator, 4℃ balance cabinet, adhesion slide, centrifuge, vortexer, laser confocal microscope, refrigerator, pseudo-vaginal sperm collector, insemination gun, etc.

[0031] In the following examples of the present invention, the sheep semen diluent was prepared by accurately weighing each reagent using an electronic balance, fully dissolving it using a magnetic stirrer, filtering and sterilizing it after dissolution, and storing it in a -20°C refrigerator.

[0032] Example 1

[0033] Sheep semen 5 times concentrated diluent, each 35.2 mL includes the following components: 0.75g soy lecithin, 2g fructose, 4.84g Tris, 0.16g bovine serum albumin, 80mg adenosine triphosphate, 100mg reduced glutathione, 2.68g citric acid, 0.5g vitamin C, 0.55g trehalose, 0.74g penicillin-streptomycin (600,000 IU), and the rest is purified water.

[0034] When in use, take the corresponding volume (V1) of 5 times concentrated dilution and incubate it in a 37°C water bath for 3 minutes, then mix it thoroughly with V2 (V2 / V1=4 / 1) volume of pure water as sheep semen diluent, sterilize it in a 72°C water bath for 30 minutes, and adjust the pH value to 7.

[0035] Comparative Example 1

[0036] Sheep yolk sperm diluent, each 176 mL includes the following components: 2 g fructose, 4.84 g Tris, 2.68 g citric acid, 0.1 g reduced glutathione, 0.16 g bovine serum albumin, 0.08 g adenosine triphosphate, 0.5 g vitamin C, 0.55 g trehalose, 0.74 g penicillin-streptomycin (600,000 IU), 40 mL yolk, and the balance is purified water. Add the above raw materials (except yolk) to purified water, stir evenly, place in a 72°C water bath for disinfection for 30 minutes, cool to room temperature, add yolk, and dilute to 176 mL with purified water, and adjust the pH to 7.

[0037] Comparative Example 2

[0038] Based on Example 1, the volume of sheep semen diluent was adjusted to 176 mL, and the remaining components and the mass of each component were the same as those in Example 1, and finally the volume was fixed with purified water. All raw materials were added to purified water, stirred evenly, and placed in a 72°C water bath for disinfection for 30 minutes, and the volume was fixed to 176 mL with purified water, and the pH value was adjusted to 7.

[0039] Comparative Example 3

[0040] Based on Comparative Example 2, the trehalose in the sheep semen diluent was replaced with 3.52 mg of proanthocyanidins, and the remaining ingredients, proportions, and methods were the same as those in Comparative Example 2.

[0041] Comparative Example 4

[0042] Based on Comparative Example 3, there is no bovine serum albumin in the sheep semen diluent, and the remaining ingredients, proportions and methods are the same as those in Comparative Example 3.

[0043] Test Example 1

[0044] Sheep semen was collected using a false vagina and the collected semen was initially examined: normal semen volume was 0.5-1.5 mL, the color was milky white or milky yellow, and there was no abnormal odor or a slight fishy odor.

[0045] After semen collection, the semen is quickly brought back to the laboratory and the quality of the collected semen is evaluated using CASA. Semen with sperm motility higher than 80%, deformity rate lower than 5%, and density greater than 1.6 billion / mL is selected and placed in a sterile centrifuge tube for verification experiments of semen diluent.

[0046] The semen was isothermally diluted using the preheated diluent of Example 1 and Comparative Examples 1-4, with the dilution multiple being 6 times; the diluted semen was sealed and placed in a 4°C refrigerator to cool and equilibrate for 4 hours; the equilibrated semen was placed in an ice-water mixture for low-temperature storage.

[0047] Motility test of sheep cryopreserved sperm: Semen stored at different times (1d, 5d, 10d, 15d) was incubated in a water bath at 37°C for 40s, and sperm kinematic parameters were tested using CASA, including total sperm motility (TM, %), linear velocity (VSL, μm / s), curvilinear velocity (VCL, μm / s), sperm head lateral swing amplitude (ALH, μm) and whipping frequency (BCF, Hz). At least 200 sperm were evaluated in each analysis, and the test results of sperm motility after sheep cryopreservation are shown in Table 1.

[0048] Table 1 Results of detection of motility parameters of sheep sperm cryopreserved

[0049]

[0050] Note: Data in the same column with different letters indicate significant differences (P < 0.05), and data with the same letters indicate no significant differences (P > 0.05)

[0051] As can be seen from Table 1, by comparing the low-temperature preservation effects of the diluents of the control example and the example, the sperm TM, VSL, VCL, ALH and BCF movement parameters of the example sperm were significantly higher than those of the control example (P < 0.05), while the sperm movement parameters of the comparison examples 1 and 2 were significantly higher than those of the comparison examples 3 and 4 (P < 0.05).

[0052] The above results show that soybean lecithin can replace egg yolk to prepare sheep semen diluent; the various components in the 5-fold concentrated sheep semen diluent of the present invention can act synergistically, and easily replacing or omitting a certain component will lead to a decrease in the semen preservation effect; and the 5-fold concentrated diluent of the present invention has a better effect on long-term low-temperature preservation of semen than ordinary diluent.

[0053] Test Example 2

[0054] Detection of plasma membrane integrity of cryopreserved sheep sperm: Semen stored at low temperature for different periods of time (1d, 5d, 10d, 15d) was repeatedly centrifuged and washed three times with PBS preheated at 37°C, and the centrifugation condition was 3000rpm for 5min. Take 30μL of semen sample for smear, dry at room temperature, add 4% paraformaldehyde to fix for 30min, and wash with PBS three times. Then stain with PI, place the slide in a wet box and incubate in the dark at 37°C for 30min, wash with PBS three times, stain with Hoechst for 10min, wash with PBS three times, press the slide in the dark, and collect images under a laser confocal microscope. Use 535-617nm ultraviolet light for excitation, blue fluorescence indicates that the plasma membrane of the sperm head is intact, and incomplete or no red fluorescence indicates that the plasma membrane of the sperm head is damaged. The results are shown in Table 2. The results of sperm plasma membrane integrity detection after 5d of cryopreservation are as follows Figure 2 shown.

[0055] Table 2 Results of plasma membrane integrity test of sheep sperm cryopreserved

[0056]

[0057] Note: Data in the same column with different letters indicate significant differences (P < 0.05), and data with the same letters indicate no significant differences (P > 0.05)

[0058] From Table 2 and Figure 2 It can be seen that by comparing these comparative examples with the diluent of the present invention, the integrity of the sperm plasma membrane of the examples is significantly higher than that of the comparative examples (P < 0.05), indicating that the 5-fold concentrated diluent of the present invention can improve the integrity of the sheep sperm plasma membrane during low-temperature storage.

[0059] Test Example 3

[0060] Detection of the acrosome integrity of sheep cryopreserved sperm: Semen stored at low temperatures for different times (1d, 5d, 10d, 15d) was repeatedly centrifuged and washed three times with PBS preheated at 37°C, and the centrifugation condition was 3000rpm for 5min. Take 30μL of semen sample for smear, dry at room temperature, add 4% paraformaldehyde to fix for 30min, and wash with PBS three times. Then stain with FITC-PNA, place the slide in a wet box and incubate in the dark at 37°C for 30min, wash with PBS three times, stain with Hoechst for 10min, wash with PBS three times, press the slide in the dark, and collect images under a laser confocal microscope. Use 494-518nm ultraviolet light for excitation, green fluorescence indicates that the sperm acrosome is intact, and incomplete green fluorescence indicates that the sperm acrosome is damaged. The results are shown in Table 3. The results of the sperm acrosome integrity test after 5 days of cryopreservation are as follows Figure 3 shown.

[0061] Table 3 Results of acrosome integrity test of sheep sperm cryopreserved

[0062]

[0063] Note: Data in the same column with different letters indicate significant differences (P < 0.05), and data with the same letters indicate no significant differences (P > 0.05)

[0064] As can be seen from Table 3, by comparing these comparative examples with the diluent of the present invention, the acrosome integrity of sperm in the examples is significantly higher than that in the comparative examples (P < 0.05), indicating that the 5-fold concentrated diluent of the present invention can improve the acrosome integrity of sheep sperm during low-temperature storage.

[0065] Test Example 4

[0066] Artificial insemination conception rate examination: 500 multiparous ewes were selected from the pasture and divided into 5 groups, and were marked respectively; artificial insemination experiments were carried out using semen stored at low temperature until the 15th day using the dilutions of the embodiment and the comparative example, and the artificial insemination conception rates were statistically shown in Table 4.

[0067] Table 4 Artificial insemination conception rate

[0068] Group Number of estrus in the same period Artificial insemination number / Estrus conception rate / % Example 1 100 98 <![CDATA[69.39(68) a ]]> Comparative Example 1 100 98 <![CDATA[65.31(64) b ]]> Comparative Example 2 100 96 <![CDATA[63.54(61) b ]]> Comparative Example 3 100 97 <![CDATA[59.79(58) c ]]> Comparative Example 4 100 96 <![CDATA[58.33(56) c ]]>

[0069] Note: Data in the same column with different letters indicate significant differences (P < 0.05), and data with the same letters indicate no significant differences (P > 0.05)

[0070] As can be seen from Table 4, by comparing the comparative example with the diluent of the present invention, the estrus conception rate of the ewes in the embodiment group is higher than that in the comparative example group (P < 0.05), indicating that the 5-fold concentrated diluent of the present invention can improve the estrus conception rate of artificial insemination and can be used for the cryopreservation of sheep semen; at the same time, the estrus conception rate of the ewes in comparative examples 1 and 2 is significantly higher than that in comparative examples 3 and 4 (P < 0.05), indicating that the various components of the 5-fold concentrated diluent of the present invention have a synergistic effect.

[0071] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A 5-fold concentrated diluent of sheep semen, characterized in that: Each 35.2 mL of concentrated diluent includes the following components by weight: 0.7-0.8 g soybean lecithin, 1.5-3 g fructose, 4.8-5.0 g Tris, 0.16-0.3 g bovine serum albumin, 60-80 mg adenosine triphosphate, 60-100 mg reduced glutathione, 2.5-2.7 g citric acid, 0.5-0.6 g vitamin C, 0.5-0.55 g trehalose, 0.7-0.8 g penicillin-streptomycin, and the rest is water.

2. The 5-fold concentrated diluent of sheep semen according to claim 1, characterized in that: Each 35.2 mL of diluent includes the following components by weight: 0.75 g soybean lecithin, 2 g fructose, 4.84 g Tris, 0.16 g bovine serum albumin, 80 mg adenosine triphosphate, 100 mg reduced glutathione, 2.68 g citric acid, 0.5 g vitamin C, 0.55 g trehalose, 0.74 g penicillin-streptomycin, and the remainder is water.

3. The 5-fold concentrated diluent of sheep semen according to claim 1, characterized in that: In each 35.2 mL of concentrated diluent, the potency of penicillin-streptomycin is 550,000-650,000 IU.

4. The method for using the 5-fold concentrated diluent of sheep semen according to any one of claims 1 to 3, characterized in that: The 5-fold concentrated sheep semen dilution was fully mixed with water and incubated at 37° C. before use.

5. The method of use according to claim 4, characterized in that: The volume ratio of the 5-fold concentrated sheep semen diluent to the water is 1:4; and the incubation time is 2-4 minutes.

6. Use of the 5-fold concentrated diluent of sheep semen according to any one of claims 1 to 3 in improving the low-temperature storage quality of sheep semen, characterized in that: The method for improving the low-temperature storage quality of sheep semen includes improving sperm motility, improving sperm plasma membrane integrity, improving sperm acrosome integrity and improving artificial insemination conception rate.

7. A method for cryopreservation of sheep semen, characterized in that: The method comprises the steps of diluting the sheep semen with the 5-fold concentrated sheep semen diluent according to any one of claims 1 to 3 and then storing the diluent at low temperature.

8. The cryopreservation method according to claim 7, characterized in that: The 5-fold concentrated sheep semen diluent is diluted with water in advance before use; the volume ratio of the 5-fold concentrated sheep semen diluent to water is 1:

4.

9. The cryopreservation method according to claim 7, characterized in that: When diluting the sheep semen, an isothermal dilution method is adopted.

10. The cryopreservation method according to claim 7, characterized in that: The sheep semen is diluted by 4-9 times.

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