An efficient antioxidant donkey sperm diluent and its preparation method and application

Abstract

The present invention relates to the technical field of antioxidant technology for livestock sperm, and provides a highly efficient antioxidant donkey sperm diluent, a preparation method thereof, and an application. A highly efficient antioxidant donkey sperm diluent includes a basal medium and additives, wherein: the basal medium contains the following components by weight in every 1000 mL of water: 30 - 60 g of glucose, 4 - 6 g of 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid, 12 - 24 g of skim milk powder, and 0.5 - 1.1 g of potassium citrate; the additives contain the following components by weight in every 1000 mL of the basal medium: 5 mg - 10 mg of sulforaphane, 0.1 mg - 0.2 mg of stigmasterol, 40 μg - 100 μg of enrofloxacin concentrate, and 3 mg - 5 mg of cefotaxime sodium. The present invention can effectively improve the conception rate of female donkeys, reduce the incidence of metritis, and improve the in vitro sperm motility.

Description

Technical Field

[0001] The present invention relates to the technical field of antioxidant technology for livestock sperm, and particularly to a highly efficient antioxidant donkey sperm diluent and its preparation method and application. Background Art

[0002] Donkeys have high economic value. Donkey skin is the main raw material for making the precious traditional Chinese medicine Ejiao, which has the effects of nourishing yin, tonifying the lungs, and enriching blood. Donkey meat is a delicious and nutritious food, which is deeply favored by consumers. With the development of China's social economy, the increase in national income, the adjustment of diet structure, and the enhancement of health preservation awareness, the demand for donkey products in domestic and foreign markets has increased rapidly, the donkey product market has been continuously expanding, and there has been a shortage of donkey resources, resulting in the continuous increase in the price of donkeys, thus bringing new vitality to the donkey breeding industry.

[0003] However, although China is a major donkey breeding country with a long history of donkey breeding, due to the late start of the development of the donkey industry in the country, the short time for the transformation of agricultural labor demand to commercial breeding, the main proportion of the breeding of meat donkeys is scattered free-range, the degree of standardized scale breeding is low, the feeding management is relatively extensive, the production method is backward, and there is no supporting breeding system, feeding standard, and feed production technology system. There is a lack of planning and design parameters for large-scale donkey farms and environmental quality control technology, resulting in relatively lagging production in the donkey breeding industry and many gaps in the field of technical research. Therefore, the development of a large-scale breeding system has become an inevitable development trend.

[0004] One of the key points in large-scale breeding is the breeding technology. Artificial insemination technology is the basis of the breeding technology and also the cornerstone of the development of the donkey industry. Donkeys are single-foal animals with a long estrus period and difficult to grasp the ovulation time. The concentrated estrus period is from March to August every year. From the manifestation of estrus in female donkeys to ovulation, it lasts for 3 - 13 days. During this period, dominant follicles continue to develop until ovulation, and the conception rate during the estrus period is 30% - 50%, and the reproductive rate is about 60%. Donkeys are animals that ejaculate into the uterus. The reproductive tract of donkeys is relatively long and the ejaculate volume is relatively large. Generally, the average ejaculate volume of male donkeys is 70 mL. If artificial insemination is carried out in large-scale donkey farms, to ensure the conception rate, the insemination volume should not be less than 200 million sperm, and the sperm motility should not be less than 80%. Therefore, developing a diluent that can extend the in vitro survival of sperm is the most fundamental.

[0005] Since the motility of sperm gradually decreases after leaving the body of male donkeys. However, the semen diluents sold on the current market have limited effects in maintaining sperm motility, and the motility of sperm significantly decreases after dilution, resulting in an inability to guarantee the conception rate. At the same time, when the vas deferens passes through the vagina, bacteria in the environment and in the vagina are easily introduced into the uterus, causing endometritis in female donkeys, which not only affects the uterine health of female donkeys but also affects embryo implantation, further affecting the conception rate. In order to improve the conception rate of female donkeys, increase the in vitro survival rate of sperm, and ensure the uterine health of female donkeys, it is particularly important to improve and optimize the formula of donkey semen diluent. Summary of the Invention

[0006] The purpose of the present invention is to provide a highly efficient antioxidant donkey sperm diluent and its preparation method and application, so as to effectively improve the conception rate of female donkeys, reduce the incidence of endometritis, and increase the in vitro motility of sperm.

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

[0008] The present invention provides a highly efficient antioxidant donkey sperm diluent, which includes a basal solution and additives, wherein:

[0009] The basal solution contains the following components by weight in every 1000 mL of water: 30 - 60 g of glucose, 4 - 6 g of 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid, 12 - 24 g of skim milk powder, and 0.5 - 1.1 g of potassium citrate;

[0010] The additives contain the following components by weight in every 1000 mL of the basal solution: 5 mg - 10 mg of sulforaphane, 0.1 mg - 0.2 mg of stigmasterol, 40 μg - 100 μg of enrofloxacin concentrate, and 3 mg - 5 mg of cefotaxime sodium.

[0011] The present invention provides a preparation method of a highly efficient antioxidant donkey sperm diluent, which includes the following steps:

[0012] (1) Mix the components of the basal solution to obtain the basal solution;

[0013] (2) Sterilize the basal solution, and add the additives under aseptic conditions to obtain the highly efficient antioxidant donkey sperm diluent.

[0014] Preferably, the sterilization conditions in step (2) are: the basal solution is maintained at a pressure of 100.4 - 106.4 kPa and a temperature of 111.3 - 131.3 °C for 15 - 30 minutes for sterilization.

[0015] Preferably, sulforaphane, stigmasterol, and enrofloxacin in the additives are dissolved in dimethyl sulfoxide and then added to the basal solution.

[0016] Preferably, the dissolution ratio of sulforaphane to dimethyl sulfoxide is 5 mg - 10 mg: 1 mL, and the dissolution ratio of stigmasterol to dimethyl sulfoxide is 0.1 mg - 0.2 mg: 100 μL.

[0017] Preferably, for the addition of enrofloxacin, 5 - 15 mg of enrofloxacin powder is dissolved in 0.5 - 1.5 mL of dimethyl sulfoxide to prepare an enrofloxacin concentrate, and then 40 μg - 100 μg of the enrofloxacin concentrate is added to 1000 mL of the base solution.

[0018] The present invention also provides an application of a highly efficient antioxidant donkey sperm diluent in the antioxidant of donkey sperm.

[0019] By adding 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid, the present invention not only ensures that the pH of the diluent is maintained at 7.5, but also provides sodium ions for sperm to ensure the normal operation of the sperm sodium - potassium pump and maintain sperm osmotic pressure; by adding potassium citrate, it not only provides citrate ions for sperm, but also provides potassium ions for sperm to ensure the normal operation of the sperm sodium - potassium pump and maintain sperm osmotic pressure.

[0020] The added sulforaphane in the present invention can effectively reduce the free radicals generated by sperm due to intense movement, inhibit lipid peroxidation reactions, protect the mitochondrial membrane and mitochondrial function, and membrane systems such as the sperm plasma membrane, and reduce sperm oxidative stress caused by factors such as radiation, drugs, toxic substances, and intense movement; the added stigmasterol in the present invention has antioxidant and anti - inflammatory effects, and can improve sperm membrane fluidity and then improve the efficiency of sperm capacitation.

[0021] The added enrofloxacin and cefotaxime sodium in the present invention can effectively inhibit the bacterial reproduction in the diluted semen, further improve the ability of sperm to survive in vitro, and play a role where 1 + 1 > 2.

[0022] At present, long - term artificial insemination in large - scale pastures is likely to cause pathogenic bacteria to develop drug resistance. Enrofloxacin is effective against severe infections caused by drug - resistant pathogenic bacteria, has no cross - drug resistance with other antibiotics, can effectively reduce the probability of female donkeys developing metritis, ensure a healthy uterine environment for female donkeys, promote the application and popularization of this diluent, and can further improve the conception rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0024] Figure 1Sperm motility test results of experimental group 1 and control group 1 in Experimental Example 1;

[0025] Figure 2 Sperm ROS test results of experimental group 1 and control group 1 at 24 h in Experimental Example 1;

[0026] Figure 3 Enrichment analysis of sperm protein phosphorylation after treating donkey semen with a highly efficient antioxidant donkey sperm diluent for experimental group 1 and control group 1 at 24 h in Experimental Example 1. Specific implementation manners

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0028] Embodiment 1

[0029] A preparation method of a highly efficient antioxidant donkey sperm diluent, comprising the following steps:

[0030] 50 g of glucose, 5.2 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 24 g of skim milk powder, and 0.8 g of potassium citrate are added with pure water to a constant volume of 1000 mL to prepare a basic solution; the basic solution is sterilized at 121.3 °C and 103.4 kPa for 25 min.

[0031] 9 mg of sulforaphane powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO), 0.1 mg of stigmasterol powder is dissolved in 100 μL of dimethyl sulfoxide (DMSO) and then added to the basic solution; 10 mg of enrofloxacin powder is dissolved in 1 mL of dimethyl sulfoxide (DMSO) to obtain an enrofloxacin concentrated solution, 100 μg is taken and added to the basic solution, and 5 mg of cefotaxime sodium is directly added to the basic solution and mixed evenly to obtain a highly efficient antioxidant donkey sperm diluent.

[0032] Embodiment 2

[0033] A preparation method of a highly efficient antioxidant donkey sperm diluent, comprising the following steps:

[0034] 60 g of glucose, 6 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 20 g of skim milk powder, and 1.1 g of potassium citrate are added with pure water to a constant volume of 1000 mL to prepare a basic solution; the basic solution is sterilized at 131.3 °C and 106.4 kPa for 30 min.

[0035] Dissolve 10 mg of sulforaphane powder in 1 mL of dimethyl sulfoxide (DMSO), dissolve 0.2 mg of stigmasterol powder in 100 μL of dimethyl sulfoxide (DMSO), and then add it to the basal solution; dissolve 15 mg of enrofloxacin powder in 1.5 mL of dimethyl sulfoxide (DMSO) to obtain a concentrated enrofloxacin solution, take 80 μg and add it to the basal solution, and directly add 4 mg of ceftiofur sodium to the basal solution and mix well to obtain a highly efficient antioxidant donkey sperm diluent.

[0036] Example 3

[0037] A preparation method of a highly efficient antioxidant donkey sperm diluent, comprising the following steps:

[0038] Prepare a basal solution by dissolving 30 g of glucose, 4 g of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid sodium salt, 12 g of skim milk powder, and 0.5 g of potassium citrate in pure water and making up the volume to 1000 mL; sterilize the basal solution at 111.3 °C and 100.4 kPa for 15 min.

[0039] Dissolve 5 mg of sulforaphane powder in 1 mL of dimethyl sulfoxide (DMSO), dissolve 0.15 mg of stigmasterol powder in 100 μL of dimethyl sulfoxide (DMSO), and then add it to the basal solution; dissolve 5 mg of enrofloxacin powder in 0.5 mL of dimethyl sulfoxide (DMSO) to obtain a concentrated enrofloxacin solution, take 40 μg and add it to the basal solution, and directly add 3 mg of ceftiofur sodium to the basal solution and mix well to obtain a highly efficient antioxidant donkey sperm diluent.

[0040] Experimental Example 1

[0041] 1. Verify that the highly efficient antioxidant donkey sperm diluent obtained in Example 1 can improve the sperm motility. The specific detection method is as follows:

[0042] Collect semen from 10 male donkeys, mix the collected semen well, and divide it into two groups on average, which are used as Experimental Group 1 and Control Group 1 respectively.

[0043] Add the semen of Experimental Group 1 to the highly efficient antioxidant donkey sperm diluent for dilution according to a volume ratio of 1:1 (undiluted semen:diluent), and add the semen of Control Group 1 to INRA82 diluent (INRA96 diluent REF.022110-022330 produced by CASO, France) for dilution according to a volume ratio of 1:1 (undiluted semen:diluent); then, place both Experimental Group 1 and Control Group 1 in the dark at 25 °C, and use a sperm motility detector to detect the sperm motility in Experimental Group 1 and Control Group 1 at 0 h, 3 h, 6 h, 12 h, and 24 h respectively. The detection results are as Figure 1 shown, by Figure 1It can be seen that with the extension of time, the sperm motility of experimental group 1 was 43.0±3.6%, which was significantly higher than that of control group 1 (29.1±5.2%). It can be seen that the highly efficient antioxidant donkey sperm diluent provided by the present invention can effectively improve sperm motility.

[0044] 2. Verify that the highly efficient antioxidant donkey sperm diluent obtained in Example 1 has a good antioxidant effect. The specific detection method is as follows:

[0045] The processes of collecting and diluting the semen of male donkeys are the same as those in Part 1. According to the results in 1, the reactive oxygen species (ROS) of experimental group 1 and control group 1 treated for 24 h were detected. Using the DCFDA / H2DCFDA-Cellular ROS Assay kit (ab113851, ABCAM, UK), the ATP concentration of sperm in the experimental group and the control group was detected according to the instructions. The results are as Figure 2 shown. The fluorescence intensities of sperm ROC detection in control group 1 and experimental group 1 were 1.51±0.04 and 0.47±0.03 respectively. The ROS content in the sperm of control group 1 was significantly higher than that in experimental group 1, indicating that the highly efficient antioxidant donkey sperm diluent obtained in Example 1 has a good antioxidant effect.

[0046] 3. Verify that the highly efficient antioxidant donkey sperm diluent obtained in Example 1 has a good antibacterial effect. The specific detection method is as follows:

[0047] Collect semen from 10 male donkeys. After mixing the semen of the 10 male donkeys collected, take two 5-mL aliquots of the mixed semen and mix them with an equal volume of a highly efficient antioxidant donkey sperm diluent and INRA82 diluent (where the INRA82 diluent contains antibiotics: penicillin 100000 IU / L and streptomycin dihydrogen 150 mg / L) respectively, serving as experimental group 2 and control group 2. Dilute experimental group 2 and control group 2 with PBS at volume ratios of 1:10, 1:100, and 1:1000 respectively. Use 5 mL of the highly efficient antioxidant donkey sperm diluent as the blank experimental group and 5 mL of the INRA82 diluent as the blank control group; then, place experimental group 2, control group 2, the blank experimental group, and the blank control group under the condition of a 37°C dark water bath, and according to the determination method of total colony count (GB4789.2-2016), determine the colony growth conditions corresponding to different times in experimental group 2, control group 2, the blank experimental group, and the blank control group. After 5 parallel experiments (the blank experimental group and the blank control group each conducted 5 groups of parallel experiments on the same sample), the detection results are shown in Table 1. By comparing experimental group 2 and control group 2, it can be seen that at the same time, the number of colonies in experimental group 2 is significantly less than that in control group 2; meanwhile, the number of colonies in the blank experimental group and the blank control group is extremely small. It can be seen that the highly efficient antioxidant donkey sperm diluent and the INRA82 diluent prepared in Example 1 are sterile themselves. Therefore, the bacteria in experimental group 2 and control group 2 are not introduced by the two diluents. It can be seen that the highly efficient antioxidant donkey sperm diluent prepared in Example 1 used in experimental group 2 has a significantly better antibacterial effect than the INRA82 diluent used in control group 2.

[0048] Table 1 Detection results of the antibacterial effects of experimental group 2 and control group 2

[0049]

[0050] 4. Verify the molecular basis for the highly efficient antioxidant donkey sperm diluent obtained in Example 1 to promote sperm in vitro survival, that is, protein phosphorylation detection. The specific detection method is as follows:

[0051] The process of collecting semen from male donkeys is the same as in part 1. According to the results in 1, conduct protein phosphorylation detection on experimental group 1 and control group 1 at 24 h. Respectively take 20 mL of experimental group 1 and control group 1, centrifuge to collect sperm, perform protein extraction, and carry out LC-MS / MS analysis for protein identification and quantification.

[0052] 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 containing Swiss-Prot and TrEMBL Equus asus reference protein sequences. The Sequest HT search engine was used with the following parameters specified: full tryptic specificity, up to 3 missed cleavages, a minimum peptide length of 6, fixed carbamidomethylation of cysteine residues (+57.02146 Da), variable modification of methionine residues by oxidation (+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 in the Orbitrap. Peptide-spectrum matches and peptides were filtered using Percolator with a false discovery rate (FDR) of less than 1%. After spectral assignment, polypeptides were assembled into proteins and further filtered based on the combined probabilities of their constituent polypeptides, with a final FDR of 1%. By default, the top-matched protein or "master protein" is the protein with the most unique peptides and the smallest percentage peptide coverage value (i.e., the longest protein). Only unique and sharp peptides were considered for quantification. The raw data was median-normalized to eliminate experimental errors. Data with more than 50% null values in the samples was filtered out. Differentially expressed protein sites met the following conditions: average fold change > 1.5 and p-value < 0.05. The protein families were analyzed using the COG (Clusters of Orthologous Groups) database. GO annotations of the proteome were from GO data (https: / / www.ebi.ac.uk / QuickGO / ). GO annotations classify proteins into three major categories: molecular function, biological process, and cellular component.

[0053] Analysis of Control Group 1 and Experimental Group 1 revealed 16 proteins with significant differences in sperm protein phosphorylation: AKAP3, AKAP4, ATP7B, ATP2A3, CCT8L2, PSMC2, VCP, ATP2B4, HSP90AA1, DNAH6, CRYBG3, SLC26A8, PRKACA, CCDC40, FSIP2, and SPA17. For example, the degradation of AKAP3 is regulated by its tyrosine phosphorylation, which in turn affects sperm capacitation; ATP2B4, also known as plasma membrane calcium ATPase 4 (PMCA4), coordinates calcium ion and nitric oxide signaling pathways to jointly regulate mouse sperm motility; SPA17, as a key protein in the cAMP / PKA signaling pathway, regulates the ROS content by controlling downstream redox signal transduction; the PSMD4 subunit was found to undergo post-translational modification during capacitation, resulting in changes in apparent molecular mass, some of which are dependent on proteasome inhibition; the content of HSP90AA1 protein in sperm can be used as a predictor of the quality of wild boar frozen semen; the EFCAB protein regulates sperm motility through dual sensing and regulation of physiological pH and calcium; the activity of protein kinase A (PRKA) is regulated by the proteasome at the beginning of human sperm capacitation; mutations in FSIP2 lead to abnormal sperm flagellum (MMAF) morphology, etc.

[0054] These proteins and other phosphorylated or dephosphorylated proteins showing differences are involved in physiological and biochemical processes such as sperm capacitation, spermatogenesis, sperm flagellar movement, ATP hydrolysis activity, and ATP binding. These proteins are distributed in regions such as the sperm fibrous sheath, the principal piece of the sperm, the midpiece of the sperm, the endpiece of the sperm, and the sperm flagellum, as Figure 3 shown. These evidences also directly demonstrate that the highly efficient antioxidant donkey sperm diluent obtained in Example 1 improves the in vitro survival ability of donkey sperm by affecting the phosphorylation or dephosphorylation of the above proteins.

[0055] 5. Verification was carried out on the good effect of the highly efficient antioxidant donkey sperm diluent obtained in Example 1 in improving the conception rate of female donkeys and reducing the incidence of endometritis. The specific detection method is as follows:

[0056] After diluting the raw sperm with the highly efficient antioxidant donkey sperm diluent obtained in Example 1 in a demonstration donkey farm, a total of 2100 inseminations were performed on female donkeys, which was used as Experimental Group 3; after diluting the raw sperm with INRA82 diluent, a total of 3200 inseminations were performed on female donkeys, which was used as Control Group 3; and the raw sperm of Experimental Group 3 and Control Group 3 was the same, the dilution ratio was the same, and the insemination position was the same. The diluted semen was all injected into the uterine horn of the female donkey.

[0057] The pregnancy status was statistically analyzed 18 days after insemination and ovulation. In experimental group 3, there were 1,321 pregnant female donkeys, with a pregnancy rate of 62.9%, and the incidence of endometritis in female donkeys was 4.4%. In control group 3, there were 1,501 pregnant female donkeys, with a pregnancy rate of 46.9%, and the incidence of endometritis in female donkeys was 12.1%.

[0058] It can be seen that compared with control group 3, the pregnancy rate in experimental group 3 has been greatly improved, while the incidence of endometritis in female donkeys has been greatly reduced. Thus, it can be seen that the diluent applicable to donkey sperm energy supply proposed by the present invention has outstanding effects in improving the pregnancy rate of female donkeys and reducing the incidence of endometritis, promotes the continuous expansion of the breeding scale, and also provides better protection for the uterine health of female donkeys.

[0059] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An efficient antioxidant donkey sperm diluent, characterized in that, It includes a basal solution and additives, wherein: The basal solution contains the following components by weight per 1000 mL of water: 30 - 60 g of glucose, 4 - 6 g of 4 - (2 - hydroxyethyl)piperazine - 1 - ethanesulfonic acid, 12 - 24 g of skim milk powder, and 0.5 - 1.1 g of potassium citrate; The additives contain the following components by weight per 1000 mL of the basal solution: 5 mg - 10 mg of sulforaphane, 0.1 mg - 0.2 mg of stigmasterol, 40 μg - 100 μg of enrofloxacin concentrate, and 3 mg - 5 mg of cefotaxime sodium; when adding the enrofloxacin concentrate, 5 - 15 mg of enrofloxacin powder is dissolved in 0.5 - 1.5 mL of dimethyl sulfoxide to prepare the enrofloxacin concentrate, and then 40 μg - 100 μg of the enrofloxacin concentrate is added to 1000 mL of the basal solution.

2. The preparation method of the high-efficiency antioxidant donkey sperm diluent according to claim 1, characterized in that, It includes the following steps: (1) Mix the components of the basal solution to obtain the basal solution; (2) Sterilize the basal solution and add the additives in a sterile environment to obtain a highly efficient antioxidant donkey sperm diluent.

3. The method according to claim 2, wherein The sterilization conditions in step (2) are: the basal solution is maintained at a pressure of 100.4 - 106.4 kPa and a temperature of 111.3 - 131.3 °C for 15 - 30 minutes for sterilization.

4. The method according to claim 3, wherein In the additives, sulforaphane and stigmasterol are respectively dissolved in dimethyl sulfoxide and then added to the basal solution.

5. The method according to claim 4, wherein The dissolution ratio of sulforaphane to dimethyl sulfoxide is 5 mg - 10 mg:1 mL, and the dissolution ratio of stigmasterol to dimethyl sulfoxide is 0.1 mg - 0.2 mg:100 μL.

6. Use of a highly efficient antioxidant donkey sperm diluent according to any one of claims 1 - 5 in the antioxidant of donkey sperm.

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