阅读说明：本技术 一种适用于驴精液的抑菌子宫角输精稀释液及其制备方法 (Bacteriostatic uterine horn sperm-infusion diluent suitable for donkey sperm and preparation method thereof ) 是由 吴海青 苏少锋 李泽廷 马跃军 孙培陪 白晋宇 高俊 于 2021-09-23 设计创作，主要内容包括：本发明公开了一种适用于驴精液的抑菌子宫角输精稀释液,还公开了一种适用于驴精液的抑菌子宫角输精稀释液的制备方法。优点在于：通过适量的添加牛磺酸、亚牛磺酸,牛血清白蛋白、肝素钠以及孕酮,可促进精子在体外获能,维持精子的活力,在对原精稀释后,仍能提高受胎率,并能实现精液高倍稀释的目的,在原精一定量的情况下,可大大提高公驴精液的利用率,进一步提高受精母驴的数量,使更多的母驴可以受胎,促进驴养殖企业养殖规模的发展壮大,提高企业的经济效益；通过适量的添加头孢西丁钠、两性霉素以及多粘菌素,可有效降低母驴发生子宫炎的几率,保证母驴健康的子宫环境,促进深角输精方式的应用推广,可为提高受胎率提供有力的保障。(The invention discloses a bacteriostatic uterine horn insemination diluent suitable for donkey semen and a preparation method of the bacteriostatic uterine horn insemination diluent suitable for donkey semen. Has the advantages that: by adding appropriate amount of taurine, hypotaurine, bovine serum albumin, heparin sodium and progesterone, the in-vitro capacitation of sperms can be promoted, the vitality of the sperms can be maintained, the conception rate can be still improved after the original sperms are diluted, the purpose of high-power dilution of the sperms can be realized, the utilization rate of the sperms of the male donkeys can be greatly improved under the condition of quantitative original sperms, the number of the fertilized female donkeys can be further improved, more female donkeys can be conception, the development of the breeding scale of the donkey breeding enterprises can be promoted, and the economic benefit of the enterprises can be improved; by adding a proper amount of cefoxitin sodium, amphotericin and polymyxin, the possibility of metritis of the donkey can be effectively reduced, the healthy uterine environment of the donkey is ensured, the application and popularization of the deep angle insemination mode are promoted, and a powerful guarantee can be provided for improving the conception rate.)

1. An antibacterial uterine horn insemination diluent suitable for donkey semen, which is characterized by comprising a solution A and a solution B, wherein:

the solution A consists of the following components: 1.2 to 1.6g of sodium chloride, 0.5 to 0.7g of monopotassium phosphate, 0.05 to 0.15g of magnesium sulfate heptahydrate, 0.3 to 0.5g of sodium bicarbonate, 4.3 to 6.1g of HEPES, 40 to 60g of glucose, 0.06 to 0.1g of sodium pyruvate, 3 to 4.2mg of sodium lactate, 12 to 24g of skimmed milk powder, 2 to 4g of taurine, 8.72 to 13.08mg of hypotaurine and 800ml of pure water;

the solution B consists of the following components: 1 to 6g of bovine serum albumin, 20 to 25mg of heparin sodium, 1.57 to 6.28mg of progesterone, 20 to 30mg of cefoxitin sodium, 20 to 30mg of amphotericin, 70 to 130mg of polymyxin and 100ml of pure water.

2. The bacteriostatic uterine horn insemination diluent suitable for donkey semen as claimed in claim 1, wherein the A solution consists of the following components: 1.52g of sodium chloride, 0.68g of monopotassium phosphate, 0.12g of magnesium sulfate heptahydrate, 0.35g of sodium bicarbonate, 4.76g of HEPES, 50g of glucose, 0.08g of sodium pyruvate, 3.6mg of sodium lactate, 24g of skimmed milk powder, 3.75g of taurine, 8.72mg of hypotaurine and 800ml of pure water.

3. The bacteriostatic uterine horn insemination diluent suitable for donkey semen as claimed in claim 1, wherein the B solution consists of the following components: bovine serum albumin 1g, heparin sodium 20mg, progesterone 3.14mg, cefoxitin sodium 24mg, amphotericin 25mg, polymyxin 100mg and pure water 100 ml.

4. A method of preparing a bacteriostatic meyer's horn insemination diluent for donkey semen as claimed in any one of claims 1 to 3, comprising the steps of:

(1) respectively taking the components of the solution A and the solution B according to the proportion, and uniformly mixing to respectively prepare the solution A and the solution B;

(2) sterilizing the solution A prepared in the step (1) at high temperature and high pressure;

(3) and (3) filtering and sterilizing the solution B prepared in the step (1) in an aseptic environment, mixing the solution B with the sterilized solution A obtained in the step (2), and adding sterile pure water to a constant volume of 1L to obtain the bacteriostatic uterine horn sperm injection diluent.

5. The preparation method of the bacteriostatic uterine horn semen deposition diluent suitable for donkey semen as claimed in claim 4, wherein in the step (2), the solution A is sterilized under the conditions of the pressure of 103.4kPa and the temperature of 121.3 ℃ for 15-30 minutes.

6. The method for preparing the bacteriostatic uterine horn insemination diluent used for donkey semen according to claim 4, wherein in the step (3), the solution B is sterilized by filtration through a 22 μm sterile filter.

The technical field is as follows:

the invention relates to the field of livestock raising, in particular to an antibacterial uterine horn sperm injection diluent suitable for donkey semen and a preparation method thereof.

Background art:

with the increasing popularity of consumers for products such as donkey meat, donkey milk, donkey gelatin and the like, the donkey breeding industry is getting stronger. In the estrus season of the donkey, the breeder can input collected donkey semen to the female donkey so as to make the female donkey to be conception and promote the reproduction of the donkey. In order to improve the conception rate of the donkey, the donkey is required to be fertilized for many times, in order to ensure the conception rate, the sperm input by each donkey is about more than 2 hundred million generally, the sperm is fertilized for 1 time every day, and the continuous insemination is required for 2-4 days, so that the conception rate can be ensured, namely, the donkey needs a great number of sperms at one end, the donkey is fatigued and softened in the late stage of the breeding season, the sperm quality is reduced, the conception rate is reduced, the development of the breeding scale of donkey breeding enterprises is seriously restricted, and the economic benefit of the enterprises is influenced. In order to increase the number of fertilized donkey mothers and enable more donkey mothers to be fertilized, at present, some donkey breeding enterprises can dilute collected donkey semen when the donkey is used for insemination. Because the motility of the sperms is gradually reduced after the sperms leave the body of the donkey, however, the semen diluent sold in the market at present only plays a role in diluting the semen, has limited effect on maintaining the motility of the sperms and does not have the function of promoting the sperm capacitation in vitro, so the conception rate cannot be guaranteed; meanwhile, in order to promote the combination of sperm and egg cells, some breeders will adopt a deep angle insemination mode, namely: semen is infused to the uterine corner of the female donkey to shorten the movement distance of the sperm, but when the vas deferens passes through the vagina, bacteria in the vagina are easily brought into the uterus, so that the female donkey can cause metritis, the uterine health of the female donkey is affected, the implantation of an embryo is affected, and the conception rate is further affected. In conclusion, in order to improve the conception rate, improve the utilization rate of the sperm of the donkey and ensure the health of the donkey, the improvement and optimization of the formula of the donkey semen diluent is very important.

The invention content is as follows:

in order to effectively improve the conception rate of the female donkey, reduce the incidence rate of metritis and promote sperm capacitation in vitro, the first aim of the invention is to provide the bacteriostatic uterine horn sperm injection diluent suitable for donkey semen, and the second aim of the invention is to provide the preparation method of the bacteriostatic uterine horn sperm injection diluent suitable for donkey semen.

The first purpose of the invention is implemented by the following technical scheme:

an antibacterial uterine horn insemination diluent suitable for donkey semen, comprising a solution A and a solution B, wherein:

the solution A consists of the following components: 1.2 to 1.6g of sodium chloride, 0.5 to 0.7g of monopotassium phosphate, 0.05 to 0.15g of magnesium sulfate heptahydrate, 0.3 to 0.5g of sodium bicarbonate, 4.3 to 6.1g of HEPES, 40 to 60g of glucose, 0.06 to 0.1g of sodium pyruvate, 3 to 4.2mg of sodium lactate, 12 to 24g of skimmed milk powder, 2 to 4g of taurine, 8.72 to 13.08mg of hypotaurine and 800ml of pure water.

The solution B consists of the following components: 1 to 6g of bovine serum albumin, 20 to 25mg of heparin sodium, 1.57 to 6.28mg of progesterone, 20 to 30mg of cefoxitin sodium, 20 to 30mg of amphotericin, 70 to 130mg of polymyxin and 100ml of pure water.

Preferably, the solution A consists of the following components: 1.52g of sodium chloride, 0.68g of monopotassium phosphate, 0.12g of magnesium sulfate heptahydrate, 0.35g of sodium bicarbonate, 4.76g of HEPES, 50g of glucose, 0.08g of sodium pyruvate, 3.6mg of sodium lactate, 24g of skimmed milk powder, 3.75g of taurine, 8.72mg of hypotaurine and 800ml of pure water.

Preferably, the solution B consists of the following components: bovine serum albumin 1g, heparin sodium 20mg, progesterone 3.14mg, cefoxitin sodium 24mg, amphotericin 25mg, polymyxin 100mg and pure water 100 ml.

The second purpose of the invention is implemented by the following technical scheme:

a preparation method of an antibacterial uterine horn insemination diluent suitable for donkey semen comprises the following steps:

(1) respectively taking the components of the solution A and the solution B according to the proportion, and uniformly mixing to respectively prepare the solution A and the solution B;

(2) sterilizing the solution A prepared in the step (1) at high temperature and high pressure;

(3) and (3) filtering and sterilizing the solution B prepared in the step (1) in an aseptic environment, mixing the solution B with the sterilized solution A obtained in the step (2), and adding sterile pure water to a constant volume of 1L to obtain the bacteriostatic uterine horn sperm injection diluent.

Preferably, in the step (2), the solution A is sterilized by maintaining the solution A under a pressure of 103.4kPa at a temperature of 121.3 ℃ for 15 to 30 minutes.

Preferably, in the step (3), the solution B is subjected to filter sterilization by using a 22 μm sterile filter.

The invention has the advantages that:

1. by adding appropriate amount of taurine, hypotaurine, bovine serum albumin, heparin sodium and progesterone, sperm capacitation in vitro can be promoted, sperm activity can be maintained, and sperm input at one time can be controlled at 2 × 10 after dilution of original sperm⁷～8×10⁷Under the condition of single dosage, the conception rate can be still improved, the aim of high-power semen dilution can be realized, the utilization rate of semen of the male donkeys can be greatly improved under the condition of the same dosage of the original semen, the quantity of the fertilized female donkeys can be further improved, more female donkeys can be subjected to conception, the development of the breeding scale of the donkey breeding enterprises is promoted, and the economic benefit of the enterprises is improved;

2. by adding a proper amount of cefoxitin sodium, amphotericin and polymyxin, the possibility of metritis of the donkey can be effectively reduced, the healthy uterine environment of the donkey is ensured, the application and popularization of the deep angle insemination mode are promoted, and a powerful guarantee can be further provided for improving the conception rate.

The specific implementation mode is as follows:

the technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

an antibacterial uterine horn insemination diluent suitable for donkey semen, comprising a solution A and a solution B, wherein:

the solution A consists of the following components: 1.52g of sodium chloride, 0.68g of monopotassium phosphate, 0.12g of magnesium sulfate heptahydrate, 0.35g of sodium bicarbonate, 4.76g of HEPES, 50g of glucose, 0.08g of sodium pyruvate, 3.6mg of sodium lactate, 24g of skimmed milk powder, 3.75g of taurine, 8.72mg of hypotaurine and 800ml of pure water.

The solution B consists of the following components: bovine serum albumin 1g, heparin sodium 20mg, progesterone 3.14mg, cefoxitin sodium 24mg, amphotericin 25mg, polymyxin 100mg and pure water 100 ml.

The effect of each component in this example is as follows:

sodium chloride is mainly used for controlling electrolyte balance in spermatids;

the potassium dihydrogen phosphate is mainly used for maintaining osmotic pressure;

magnesium ions in magnesium sulfate heptahydrate contribute to sperm motility;

HEPES is a hydrogen ion buffer, can control a constant pH range for a long time, and has strong buffering capacity between pH7.2 and 7.4. Because the semen is alkalescent, the pH value is 7.2-7.8; thus, HEPES has a better effect on maintaining sperm pH.

Sodium lactate can improve antioxidant effect and is used for regulating pH;

the skim milk powder has better buffering effect because the milk is a natural buffering agent; however, milk fat restricts sperm movement, and casein in the milk can maintain sperm motility. Therefore, the skim milk powder is more convenient to use than the skim milk.

Glucose is the main energy source substance for sperm capacitation and maintaining super-activation movement, and is helpful for donkey sperm capacitation;

the sodium pyruvate in the formula mainly plays a strong antioxidation role, improves the tolerance of sperms under the anoxic condition, and can be consumed under the anoxic or aerobic condition through the systemic alkalization effect to protect the basic functions of sperms.

The sodium bicarbonate also plays an important role in maintaining osmotic pressure, the sperms are in isotonic seminal plasma in vivo, if the salt concentration of the seminal plasma part is higher, the osmotic pressure is high, and the sperms are easy to dehydrate; on the contrary, the low osmotic pressure is easy to swell the sperms, and the proper amount of sodium bicarbonate can effectively maintain the osmotic pressure of the donkey semen diluent.

Progesterone can induce donkey sperm to generate acrosome reaction, and the acrosome reaction marks the completion of sperm capacitation;

the bovine serum albumin can improve the sperm motility and the survival rate and promote the sperm capacitation;

taurine, hypotaurine, and heparin sodium have positive effects in sperm capacitation in vitro and maintaining sperm motility.

Meanwhile, the bacteria generally causing metritis mainly include: escherichia coli, Staphylococcus, Pseudomonas aeruginosa, Bacillus, and Candida. The cefoxitin sodium has stronger antibacterial action on sensitive strains of staphylococcus aureus, streptococcus pneumoniae or other streptococcus (except enterococcus) in gram-positive bacteria; amphotericin is used for treating visceral or systemic infection caused by cryptococcus, coccidioidomycosis, histoplasma capsulatum, blastomyces, sporothrix, candida, mucor, aspergillus, etc.; polymyxin (polymyxin) has antibacterial effect on Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae. Therefore, cefoxitin sodium, amphotericin and polymyxin have positive effects on deep horn insemination and infection resistance.

Example 2:

a preparation method of an antibacterial uterine horn insemination diluent suitable for donkey semen comprises the following steps:

(1) respectively taking the components of the solution A and the solution B according to the proportion, and uniformly mixing to respectively prepare the solution A and the solution B;

(2) maintaining the solution A prepared in the step (1) for 15-30 minutes under the conditions of the pressure of 103.4kPa and the temperature of 121.3 ℃, and carrying out sterilization treatment;

(3) filtering and sterilizing the solution B prepared in the step (1) by using a 22-micron sterile filter under a sterile environment, mixing the solution B with the sterilized solution A obtained in the step (2), and adding sterile pure water to a constant volume of 1L to obtain the bacteriostatic uterine horn sperm injection diluent.

Since the bovine serum albumin, cefoxitin sodium, amphotericin, and polymyxin in the solution B are active substances and the functions thereof are destroyed by high temperature, the solution B in this embodiment is filtered and sterilized only by a sterile filter.

Comparative example 1:

in order to verify that the bacteriostatic uterine horn sperm injection diluent suitable for donkey semen provided by the invention can improve the activity of sperms, the comparative example detects the activity of sperms in semen diluted by the bacteriostatic uterine horn sperm injection diluent prepared in the example 2, and the specific detection method comprises the following steps:

semen is collected from 10 male donkeys, and the collected semen is evenly mixed and evenly divided into two groups which are respectively used as an experimental group 1 and a control group 1. Adding the bacteriostatic uterine horn insemination diluent into the semen of the experimental group 1 according to the volume ratio of 1:1 (original semen: diluent) for dilution, and adding the INRA82 diluent into the semen of the control group 1 according to the volume ratio of 1:1 (original semen: diluent) for dilution; then, placing the experimental group 1 and the control group 1 in a dark water bath condition at 37 ℃, and detecting the activity of the sperms in the experimental group 1 and the control group 1 by using a sperm activity detector at 0h, 3h, 6h, 9h and 24h respectively, wherein the detection results are shown in table 1;

TABLE 1 sperm motility test results of experiment group 1 and control group 1

Activity (%)	0h	3h	6h	9h	24h
						Experimental group 1	71.6	64.3	52.7	43.5	18.4
Control group 1	71.6	59.5	46.6	39.8	11.1

As can be seen from the table 1, the sperm motility of the experimental group 1 is obviously higher than that of the control group 1 along with the prolonging of the time, and the bacteriostatic sperm injection diluent for uterine horn, which is provided by the invention and is suitable for donkey semen, can effectively improve the sperm motility.

Comparative example 2:

in order to verify that the bacteriostatic uterine horn sperm injection diluent suitable for donkey semen provided by the invention has a good bacteriostatic effect, the comparative example detects the bacteriostatic effect of semen diluted by the bacteriostatic uterine horn sperm injection diluent prepared in example 2, and the specific detection method comprises the following steps:

semen is collected from 10 male donkeys, and after the collected semen is mixed evenly, 5ml of semen is respectively taken and mixed with the bacteriostatic uterine horn insemination diluent and INRA82 diluent (containing antibiotics: 100000IU/L penicillin and 150mg/L streptomycin dihydro) in equal volume respectively to be used as an experimental group 2 and a control group 2 respectively. The experimental group 2 and the control group 2 were diluted with PBS at a volume ratio of 1:10, 1:100, 1:1000, respectively. 5ml of the bacteriostatic uterine horn sperm injection diluent prepared by the invention is used as a blank experimental group, and 5ml of INRA82 diluent is used as a blank control group; then, the experimental group 2, the control group 2, the blank experimental group and the blank control group are placed under a 37 ℃ dark water bath condition, the growth conditions of bacterial colonies in the experimental group 2, the control group 2, the blank experimental group and the blank control group corresponding to three dilution gradients at different times are determined according to a bacterial colony total number determination (GB4789.2-2016) determination method, and after multiple experiments, the detection results are shown in table 2, the experimental group 2 shows a strong bacteriostatic effect under different dilution gradients, and particularly the bacteriostatic effect is optimal within 24 h. In Table 2, "- -" indicates that the colonies were full and could not be counted.

TABLE 2 results of the test group 2 and the control group 2 for the bacteriostatic effect

Since bacteria may be introduced during the experiment, the comparative example performed three experiments on the same sample for the blank experimental group and the blank control group, respectively, in order to ensure the reliability of experimental data.

As can be seen from Table 2, the colony count of the experimental group 2 is obviously less than that of the control group 2 at the same time under different dilution gradients by comparing the experimental group 2 with the control group 2; meanwhile, the numbers of colonies in the blank experimental group and the blank control group were very small, and it can be seen that the bacteriostatic uterine horn insemination diluent and the INRA82 diluent prepared by example 2 were sterile themselves, so that the bacteria in the experimental group 2 and the control group 2 were not carried by the two diluents. It can be seen that the bacteriostatic uterine horn insemination diluent prepared in example 2 adopted in the experimental group 2 has a significant bacteriostatic effect compared with the INRA82 diluent adopted in the control group 2.

Comparative example 3:

in order to verify that the bacteriostatic meurospermum diluent suitable for the donkey semen has good effects on improving the conception rate of the female donkey and reducing the incidence rate of metritis, the comparative example shows that after the bacteriostatic meurospermum diluent prepared by the invention is used for diluting the original semen in a project cooperation demonstration donkey farm, 1280 heads of secondary female donkeys are subjected to semen deposition in total and serve as an experimental group 3; diluting original semen with INRA82 diluent to give total semen deposition of 5400 times of donkey, and using as control group 3; and the original semen of the experimental group 3 and the original semen of the control group 3 are the same, the dilution ratio is the same, and the semen deposition positions are the same, namely the diluted semen is deposited at the uterine angle of the donkey.

The conception condition is counted by pregnancy test 18 days after insemination and ovulation, in the experimental group 3, the conception rate of the conception donkey is 764 heads, 59.7 percent and the incidence rate of the mother donkey metritis is 4.3 percent. In the control group 3, the number of the pregnant donkey was 2432, the pregnancy rate was 45%, and the incidence rate of hysteritis of the donkey was 10.8%.

Therefore, compared with the control group 3, the experimental group 3 has the advantages that the conception rate is greatly improved, the hysteritis incidence rate of the female donkey is greatly reduced, and therefore, the bacteriostatic uterine horn insemination diluent suitable for donkey semen provided by the invention has outstanding effects on improving the conception rate of the female donkey and reducing the hysteritis incidence rate, promotes the continuous expansion of the culture scale, and is more guaranteed for the uterine health of the female donkey.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.