A diluent for promoting donkey sperm energy supply, and its preparation method and application

Abstract

The present invention relates to the technical field of livestock sperm energy supply, and provides a diluent for promoting donkey sperm energy supply, as well as its preparation method and application. A diluent for promoting donkey sperm energy supply, comprising a base liquid and an additive, wherein: the base liquid comprises the following components by weight per 1000mL of water: 30-60g of glucose, 4-6g of sodium 4-hydroxyethylpiperazineethanesulfonate, 12-24g of skimmed milk powder, and 0.5-1.1g of potassium citrate; the additive comprises the following components by weight per 1000mL of base liquid: 200-800mg of NADH, 40-100μg of enrofloxacin concentrate, and 3-5mg of ceftiofur sodium. By adding an appropriate amount of enrofloxacin and ceftiofur sodium, the present invention can effectively reduce the chance of metritis in female donkeys, ensure a healthy uterine environment for female donkeys, promote the application and promotion of deep-angle insemination methods, and provide a strong guarantee for improving conception rates.

Description

Technical Field

[0001] The invention relates to the technical field of livestock sperm energy supply and provides a diluent for promoting donkey sperm energy supply, a preparation method and an application thereof. Background Art

[0002] Donkeys have a high economic value. Donkey hide is the primary raw material for the precious Chinese medicine donkey-hide gelatin, which nourishes yin, tonifies the lungs, and replenishes blood. Donkey meat is a delicious and nutritious food favored by consumers. With my country's socioeconomic development and rising national income, coupled with dietary adjustments and a growing awareness of health and wellness, demand for donkey products has surged both domestically and internationally. The market for donkey products continues to expand, leading to a shortage of donkey resources and a continuous rise in prices, bringing new vitality to the donkey farming industry.

[0003] However, although my country is a major donkey-raising country with a long history of donkey breeding, the donkey industry started late in the country and the time for agricultural demand to shift to commercial breeding is relatively short. The breeding industry of meat donkeys is mainly based on scattered and free-range breeding, with a low degree of standardized and large-scale breeding, extensive breeding management, and backward production methods. A supporting breeding system, feeding standards, and feed production technology system have not yet been formed. There is a lack of supporting planning and design parameters and environmental quality control technologies for large-scale donkey farms, resulting in relatively lagging donkey industry production. There are still many gaps in the field of technical research, so the development of a large-scale breeding system has become an inevitable development trend.

[0004] A key element of large-scale breeding is reproductive technology. Artificial insemination is its foundation and has become the cornerstone of the donkey industry's development. Donkeys are single-birthing animals with a long estrus period, making ovulation difficult to time. The peak estrus period runs from March to August each year, lasting 3-13 days from the onset of estrus in female donkeys to ovulation. During this period, the dominant follicle continues to develop until ovulation. The conception rate during estrus is 30%-50%, and the reproductive rate is around 60%. Donkeys ejaculate intrauterine, and their reproductive tracts are long, resulting in large ejaculatory volumes. The average male ejaculate volume is 70 mL. If large-scale donkey farms implement artificial insemination, the insemination volume must be at least 200 million sperm, and sperm motility must not fall below 80%. Therefore, developing a diluent to prolong sperm survival in vitro is essential.

[0005] Sperm motility gradually decreases after leaving the male donkey's body. However, currently available semen extenders are limited in their effectiveness at maintaining sperm motility, significantly reducing sperm motility after dilution, leading to uncertain conception rates. Furthermore, as the vas deferens passes through the vagina, it can easily introduce bacteria from the environment and the vagina into the uterus, potentially causing metritis in female donkeys. This not only affects uterine health but also hinders embryo implantation, further impacting conception rates. Therefore, in order to improve conception rates, enhance sperm survival in vitro, and ensure uterine health in female donkeys, it is crucial to refine and optimize the formula of donkey semen extenders. Summary of the Invention

[0006] The purpose of the present invention is to provide a diluent for promoting the energy supply of donkey sperm, a preparation method and an application thereof, so as to effectively improve the conception rate of female donkeys, reduce the incidence of metritis and improve the sperm motility in vitro.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a diluent for promoting the energy supply of donkey sperm, comprising a base liquid and an additive, wherein:

[0009] The base solution includes the following ingredients by weight per 1000 mL of water: 30-60 g of glucose, 4-6 g of sodium 4-hydroxyethylpiperazineethanesulfonate, 12-24 g of skimmed milk powder, and 0.5-1.1 g of potassium citrate;

[0010] The additive comprises the following components by weight per 1000 mL of the base solution: 200-800 mg of NADH, 40-100 μg of enrofloxacin concentrated solution, and 3-5 mg of ceftiofur sodium.

[0011] The present invention provides a method for preparing a diluent for promoting the energy supply of donkey sperm, comprising the following steps:

[0012] (1) mixing the components of the base liquid to obtain the base liquid;

[0013] (2) Sterilize the base liquid and add the additive under a sterile environment to obtain a diluent that promotes the energy supply of donkey sperm.

[0014] Preferably, the sterilization conditions in step (2) are: the base liquid is sterilized under the conditions of a pressure of 100.4 to 106.4 kPa and a temperature of 111.3 to 131.3° C. for 15 to 30 minutes.

[0015] Preferably, when adding enrofloxacin, 5-15 mg of enrofloxacin powder is dissolved in 0.5-1.5 mL of dimethyl sulfoxide to prepare an enrofloxacin concentrated solution, and then 40 μg-100 μg of the enrofloxacin concentrated solution is added to 1000 mL of the base solution.

[0016] The present invention also provides an application of a diluent for promoting donkey sperm energy supply in sperm energy supply.

[0017] The present invention can promote the energy supply for sperm motility in vitro, promote ATP production, and maintain sperm motility by adding NADH; NADH also has a strong antioxidant ability, can react with free radicals, inhibit lipid peroxidation, protect mitochondrial membranes and mitochondrial functions, as well as membrane systems such as sperm plasma membranes, and can also reduce sperm oxidative stress caused by factors such as radiation, drugs, toxic substances, and strenuous exercise.

[0018] The sodium 4-hydroxyethylpiperazine ethanesulfonate added in the present invention not only ensures that the pH value of the diluent is maintained at 7.5, but also provides sodium ions for sperm, thereby ensuring the normal operation of the sperm sodium-potassium pump and maintaining the sperm osmotic pressure. The addition of potassium citrate not only ensures that citrate ions are provided to sperm, but also provides potassium ions for sperm, thereby ensuring the normal operation of the sperm sodium-potassium pump and maintaining the sperm osmotic pressure.

[0019] The enrofloxacin and ceftiofur sodium added in the present invention can effectively inhibit bacterial reproduction in diluted semen, further improve the ability of sperm to survive in vitro, and play the role of 1+1 being greater than 2.

[0020] At present, long-term artificial insemination in large-scale ranches can easily cause pathogens to develop drug resistance. Enrofloxacin is effective against serious infections caused by drug-resistant pathogens and has no cross-resistance with other antibiotics. It can effectively reduce the chance of metritis in female donkeys, ensure a healthy uterine environment for female donkeys, promote the application and promotion of this diluent, and further improve the conception rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0022] Figure 1 The sperm motility test results of experimental group 1 and control group 1 in experimental example 1;

[0023] Figure 2 The sperm ATP test results of experimental group 1 and control group 1 at 24 hours in experimental example 1;

[0024] Figure 3 This is the enrichment analysis of sperm protein phosphorylation after donkey sperm was treated with the energy diluent of experimental group 1 and control group 1 in Experimental Example 1 at 24 hours. DETAILED DESCRIPTION

[0025] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Example 1

[0026] A method for preparing a diluent for promoting donkey sperm energy supply comprises the following steps:

[0027] A base solution was prepared by adding purified water to 49 g of glucose, 5.2 g of sodium 4-hydroxyethylpiperazineethanesulfonate, 24 g of skimmed milk powder, and 0.8 g of potassium citrate to 1000 mL. The base solution was sterilized at 121.3° C. and 103.4 kPa for 25 min.

[0028] 400 mg of NADH and 5 mg of ceftiofur sodium were directly added to the base solution and mixed evenly. 10 mg of enrofloxacin powder was dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain an enrofloxacin concentrated solution. 100 μg of the solution was added to the base solution to obtain a diluent for promoting donkey sperm energy supply. Example 2

[0029] A method for preparing a diluent for promoting donkey sperm energy supply comprises the following steps:

[0030] A base solution was prepared by adding purified water to 60 g of glucose, 6 g of sodium 4-hydroxyethylpiperazineethanesulfonate, 20 g of skim milk powder, and 1.1 g of potassium citrate to a volume of 1000 mL. The base solution was sterilized at 131.3° C. and 106.4 kPa for 30 min.

[0031] 800 mg of NADH and 4 mg of ceftiofur sodium were directly added to the base solution and mixed evenly. 15 mg of enrofloxacin powder was dissolved in 1.5 mL of dimethyl sulfoxide (DMSO) to obtain an enrofloxacin concentrated solution. 80 μg was added to the base solution to obtain a diluent for promoting donkey sperm energy supply. Example 3

[0032] A method for preparing a diluent for promoting donkey sperm energy supply comprises the following steps:

[0033] A base solution was prepared by adding 30 g of glucose, 4 g of sodium 4-hydroxyethylpiperazineethanesulfonate, 12 g of skimmed milk powder, and 0.5 g of potassium citrate to 1000 mL with purified water; the base solution was sterilized at 111.3° C. and 100.4 kPa for 15 min.

[0034] 200 mg of NADH and 3 mg of ceftiofur sodium were directly added to the base solution and mixed evenly. 5 mg of enrofloxacin powder was dissolved in 0.5 mL of dimethyl sulfoxide (DMSO) to obtain an enrofloxacin concentrated solution. 40 μg was added to the base solution to obtain a diluent for promoting donkey sperm energy supply.

[0035] Experimental Example 1

[0036] 1. The diluent for promoting the energy supply of donkey sperm obtained in Example 1 can be used to verify that it can improve the motility of sperm. The specific detection method is as follows:

[0037] Semen was collected from 10 male donkeys, and the collected semen was mixed and evenly divided into two groups, namely experimental group 1 and control group 1.

[0038] The semen of the experimental group 1 was diluted by adding donkey sperm energy diluent at a volume ratio of 1:1 (original semen: diluent), and the semen of the control group 1 was diluted by adding INRA82 diluent (INRA96 diluent REF.022110-022330 produced by France Cassu Company) at a volume ratio of 1:1 (original semen: diluent); then, both the experimental group 1 and the control group 1 were placed in the dark at 25°C, and the sperm motility of the experimental group 1 and the control group 1 was tested using a sperm motility tester at 0h, 3h, 6h, 12h, and 24h, respectively. The test results are shown as follows: Figure 1 As shown by Figure 1 It can be seen that as time goes by, the sperm motility of experimental group 1 is 48.0±5.8%, which is significantly higher than 29.1±7.8% of control group 1. It can be seen that the diluent suitable for donkey sperm energy supply proposed by the present invention can effectively improve sperm motility.

[0039] 2. The diluent for promoting the energy supply of donkey sperm obtained in Example 1 was verified to have a good energy supply effect. The specific detection method is as follows:

[0040] The process of collecting and diluting male donkey semen was the same as in Part 1. Based on the results in Part 1, the total sperm cell ATP concentration of the experimental group 1 and the control group 1 was tested for 24 hours. The ATP content detection kit (S0026, Beyotime, Nanjing, China) was used to detect the sperm ATP concentration of the experimental group and the control group according to the instructions. 100 μl of ATP test solution was added to all wells and incubated at room temperature for 5 minutes to consume background ATP. 100 μl of sample or standard solution was added to the wells, mixed quickly with a pipette, and placed in a UV spectrophotometer for detection. Figure 2 As shown, the ATP concentration of each sample was calculated according to the standard curve, and the ATP concentrations in control group 1 and experimental group 1 were respectively 2.7±0.7nmol / 10 7 and 4.1±0.4nmol / 10 7It can be seen that the diluent for promoting the energy supply of donkey sperm provided by the present invention has a good energy supply effect.

[0041] 3. The diluent for promoting the energy supply of donkey sperm obtained in Example 1 has a good antibacterial effect and is verified by the following specific detection method:

[0042] Semen was collected from 10 male donkeys and mixed thoroughly. Two 5 mL portions of semen were taken and mixed with equal volumes of sperm energy-boosting diluent and INRA82 diluent (INRA82 diluent also contains antibiotics: penicillin 100,000 IU / L and streptomycin dihydrochloride 150 mg / L). These were used as experimental group 2 and control group 2, respectively. The experimental group 2 and control group 2 were diluted with PBS at a volume ratio of 1:10, 1:100, and 1:1000, respectively. 5 mL of donkey sperm energy diluent was used as a blank experimental group, and 5 mL of INRA82 diluent was used as a blank control group; then, the experimental group 2, the control group 2, the blank experimental group, and the blank control group were placed in a light-proof water bath at 37 ° C. According to the total colony count determination method (GB4789.2-2016), the colony growth corresponding to the experimental group 2, the control group 2, the blank experimental group, and the blank control group at different times was determined. After 5 parallel experiments (the blank experimental group and the blank control group were subjected to 5 parallel experiments on the same sample), the test results are shown in Table 1. By comparing the experimental group 2 and the control group 2, it can be seen that at the same time, the number of colonies in the experimental group 2 was significantly less than that in the control group 2; at the same time, the number of colonies in the blank experimental group and the blank control group was very small. It can be seen that the sperm energy diluent and the INRA82 diluent prepared in Example 1 are sterile themselves, so the bacteria in the experimental group 2 and the control group 2 are not brought in by the two diluents. It can be seen that the sperm energy diluent prepared in Example 1 used in experimental group 2 has a more significant antibacterial effect than the INRA82 diluent used in control group 2.

[0043] Table 1 Antibacterial effect test results of experimental group 2 and control group 2

[0044]

[0045]

[0046] 4. The molecular mechanism of promoting sperm motility of the diluent obtained in Example 1 for promoting donkey sperm energy supply was verified, i.e., protein phosphorylation detection. The specific detection method is as follows:

[0047] The procedure for collecting semen from male donkeys was the same as in Part 1. Based on the results in Part 1, protein phosphorylation was assayed in experimental group 1 and control group 1 after 24 hours. Sperm from each of experimental group 1 and control group 1 was collected by centrifugation, protein extraction was performed, and LC-MS / MS analysis was performed for protein identification and quantification. RAW files were analyzed using the Proteome Discoverer suite (version 2.4, Thermo Fisher Scientific). MS2 spectra were searched against the UniProtKB Equus asus proteome database, which contains Swiss-Prot and TrEMBL Equus asus reference protein sequences. The Sequest HT search engine was used with the following parameters: full tryptic specificity, a maximum of three missed cleavages, a minimum peptide length of six, fixed carbamidine methylation of cysteine ​​residues (+57.02146 Da), variable modifications by oxidation of methionine residues (+15.99492 Da), and phosphorylation of serine, threonine, and tyrosine (79.966 Da), a precursor mass tolerance of 15 ppm, and a fragment mass tolerance of 0.02 Da for MS2 spectra collected on an Orbitrap. Percolator was used to filter peptide spectral matches and peptides with a false discovery rate (FDR) of less than 1%. After spectral assignment, peptides were assembled into proteins and further filtered based on the combined probability of their constituent peptides, with a final FDR of 1%. By default, the top matching protein, or "master protein," was the protein with the most unique peptides and the lowest percent peptide coverage value (i.e., the longest protein). Only unique and sharp peptides were considered for quantification. Raw data were median-normalized to eliminate experimental variation. Data with more than 50% null values ​​were filtered out. Differentially expressed protein sites met the following criteria: mean ratio change > 1.5 and p-value < 0.05. Protein families were analyzed using the COG (Clusters of Orthologous Groups) database. The GO annotation of the proteome was obtained from the GO database (https: / / www.ebi.ac.uk / QuickGO / ). GO annotation classifies proteins into three categories: molecular function, biological process, and cellular component.

[0048] Analysis of control group 1 and experimental group 1 revealed significant differences in the phosphorylation of 13 sperm proteins: NUP35, SRRT, MLF1, PDE8A, SLC26A8, PRKACA, AKAP3, AKAP4, FSIP2, CRYBG3, EFCAB6, EFCAB5, and CAPN11. For example, AKAP3 degradation is regulated by its tyrosine phosphorylation, thereby affecting sperm capacitation; CAPN11 is involved in regulating calcium-dependent signal transduction in sperm; EFCAB proteins regulate sperm motility through dual sensing of physiological pH and calcium; protein kinase A (PRKA) activity is regulated by the proteasome at the onset of human sperm capacitation; and mutations in FSIP2 can lead to abnormal sperm flagella (MMAF) morphology.

[0049] These proteins and other proteins that show differential phosphorylation or dephosphorylation are involved in sperm capacitation, protein kinase A binding, calcium ion binding and other physiological and biochemical processes of sperm cells. These proteins are distributed in the main segment, mid-sperm, terminal end and sperm fiber sheath, such as Figure 3 These evidences also directly indicate that the donkey sperm energy diluent obtained in Example 1 can effectively promote the phosphorylation or dephosphorylation of sperm-related proteins and directly participate in the physiological and biochemical activities of sperm.

[0050] 5. The diluent for promoting the energy supply of donkey sperm obtained in Example 1 has good effects on improving the conception rate of female donkeys and reducing the incidence of metritis. The specific detection method is as follows:

[0051] In the demonstration donkey farm, after the original semen was diluted with the sperm-energized diluent obtained in Example 1, a total of 2,300 female donkeys were inseminated, and this was used as experimental group 3; after the original semen was diluted with INRA82 diluent, a total of 3,400 female donkeys were inseminated, and this was used as control group 3; and the original semen of experimental group 3 and control group 3 was the same, with the same dilution ratio and the same insemination position, that is, the diluted semen was inseminated into the uterine horn of the female donkey.

[0052] Eighteen days after insemination and ovulation, pregnancy tests were performed to assess conception rates. In experimental group 3, 1,490 sows conceived, with a conception rate of 64.8% and a metritis incidence rate of 4.5%. In control group 3, 1,612 sows conceived, with a conception rate of 47.4% and a metritis incidence rate of 11.5%.

[0053] It can be seen that the conception rate of experimental group 3 was greatly improved compared with control group 3, while the incidence of metritis in female donkeys was greatly reduced. It can be seen that the diluent suitable for donkey sperm energy supply proposed by the present invention has outstanding effects in improving the conception rate of female donkeys and reducing the incidence of metritis, promoting the continuous expansion of breeding scale, and better protecting the uterine health of female donkeys.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A diluent for promoting the energy supply of donkey sperm, characterized in that: Includes base fluid and additives, including: The base solution includes the following ingredients by weight per 1000 mL of water: 30-60 g of glucose, 4-6 g of sodium 4-hydroxyethylpiperazineethanesulfonate, 12-24 g of skim milk powder, and 0.5-1.1 g of potassium citrate; The additive includes the following components by weight per 1000 mL of the base solution: 200-800 mg of NADH, 40-100 μg of enrofloxacin concentrated solution, and 3-5 mg of ceftiofur sodium.

2. A method for preparing the diluent for promoting donkey sperm energy supply according to claim 1, characterized in that: The steps include: (1) mixing the components of the base liquid to obtain the base liquid; (2) Sterilize the base liquid and add the additive under a sterile environment to obtain a diluent that promotes the energy supply of donkey sperm.

3. The preparation method according to claim 2, wherein The sterilization conditions of step (2) are as follows: the base liquid is sterilized at a pressure of 100.4-106.4 kPa and a temperature of 111.3-131.3°C for 15-30 minutes.

4. The preparation method according to claim 3, wherein When adding enrofloxacin, 5-15 mg of enrofloxacin powder is dissolved in 0.5-1.5 mL of dimethyl sulfoxide to prepare an enrofloxacin concentrated solution, and then 40 μg-100 μg of the enrofloxacin concentrated solution is added to 1000 mL of the base solution.

5. Use of a diluent for promoting donkey sperm energy supply according to any one of claims 1 to 4 in sperm energy supply.

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