CN101554152B - Frozen semen diluent and preparation method thereof - Google Patents

Abstract

The invention discloses a novel frozen semen diluent for improving the quality and fertilization rate of frozen semen of cattle after freezing and thawing and a preparation method thereof. The prepared frozen semen diluent contains: sodium citrate: 0.9-1.2 g, sucrose: 3-5 g, egg yolk: 18-22 ml, glycerol: 4-7 ml, vitamin E: 6-9 mg, vitamin C: 500-700 mg, spectinomycin: 35 mg, and gentamicin 80,000 units per 100 ml. The invention adds _VE and _VC to the semen diluent for the first time, which can significantly improve the sperm viability and acrosome integrity rate after freezing and the activity of antioxidant enzymes in seminal plasma, thereby improving the sperm quality and fertilization rate after freezing and thawing.

Description

A kind of frozen semen diluent and preparation method thereof

technical field

The invention belongs to the technical category of improving bovine artificial insemination, and in particular relates to a frozen semen diluent used for bovine frozen semen to improve the quality of bovine sperm after freezing and a preparation method thereof.

Background technique

The successful freezing of domestic animal semen has had a profound impact on the development of artificial insemination technology. Firstly, it has laid the foundation for expanding the utilization rate and prolonging the utilization time of excellent breeding males. Secondly, it has provided a broad scope for the development of artificial insemination and low temperature biology space. Because frozen semen is more convenient in transportation, no matter in terms of time or space, the mating efficiency of male animals can be fully exerted, and the pace of livestock breed improvement has been accelerated. Although all countries have launched research on frozen semen of cattle, pigs, horses, and chickens, only the artificial insemination technology of frozen bovine semen is widely used in production practice. In the development of dilutions of livestock frozen semen, the main functions of all dilutions and storage environments are to provide enough nutrients for sperm metabolism, to prevent damage caused by rapid freezing of sperm, to buffer to prevent pH changes, and to maintain Appropriate osmotic pressure, balance electrolytes, inhibit bacterial growth, expand the dilution factor to increase the number of inseminations, improve the survival rate of sperm after freezing, and improve the utilization efficiency of fine-bred livestock and semen.

In recent years, with the development of my country's economy and the improvement of people's living standards, milk has gradually entered thousands of households, so the number of dairy cows has grown rapidly in my country in recent years. However, in our country, the high-yielding dairy cows are not well-bred, the herd size is small, and the low yield has become a factor restricting the development of dairy cows. Therefore, it has become urgent to use the frozen semen of high-yielding bulls to improve low-yielding dairy cows in my country.

In order to continuously improve the freezing effect of dairy cow frozen semen, in the past 60 years, experts at home and abroad have been working hard to explore and find the most suitable cryoprotectant to improve the freezing effect of dairy cow and beef cattle frozen semen. Studies at home and abroad have shown that sperm DNA damage intensifies after freezing-thawing treatment, resulting in reduced sperm quality and fertilization rate after freezing and thawing sperm, which has always been a problem that plagues sperm fertilization ability. Therefore, the quality of frozen semen has become an important factor restricting the success rate of artificial insemination of dairy cows and beef cattle. However, in production practice, there are often low vigor and low fertilization rate of sperm after freezing and thawing, which affects the effect of artificial insemination. Therefore, according to the current trend, use laboratory conditions to study the formula of dairy cow semen diluent and develop a new semen diluent, which can not only improve the survival rate and fertilization rate of sperm after freezing and thawing, but also speed up the development of dairy cows in my country. It plays a positive role in rapidly improving the improvement and milk production of dairy cows, increasing the economic benefits of the dairy industry, and stimulating local and regional economic development.

The following are the relevant references searched by the applicant:

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[6] Luo Hailing, Jia Zhihai, Zhu Shien, etc., Effect of Adding Vitamin E in the Diluent on the Quality of Sheep Frozen Semen [J]. Chinese Journal of Animal Husbandry, 2004, 40(11): 7-10.

[7] B.A.Ball, V.Medina, C.G.Gravance, I.Baumber.Effect of antixidants on preservation of motity viability and acrosomalintegrity of equine spermatozoa during storage at 5℃, Theriogenology2001, 56:577-569.

[8] Nabil Mansour, Mary A. McNiven, Gavin F. Richardson. The effect of dietary supplementation with blueberry, a-tocopherol or astaxanthinon oxidative stability of Arctic char (Salvelinus alpinus) semen. Theriogenology 66 (2006) 373-382.

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[14]Ukeda H, Maeda S, Ishii T, Sawamura M. Spectrophotometric assay for superoxide dismutase based on tetrazolium salt3'-{1-[(Phenylamino)-carbonyl]-34-tetrazolium}-bis(4-methoxy-6-nitro ) benzenesulphonic acid hydrate reduction by xanthinexanthineoxidase. Anal Biochem 1997; 251: 206-209.

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Contents of the invention

In view of the defects or deficiencies in the above-mentioned prior art, the object of the present invention is to provide a novel frozen semen diluent and its preparation method for improving the quality and fertilization rate of bovine frozen semen after freezing and thawing. The prepared frozen semen diluent can significantly increase the survival rate and acrosome integrity rate of sperm after freezing and the activity of antioxidant enzymes in seminal plasma, thereby improving the quality and fertilization rate of sperm after freezing and thawing.

In order to realize above-mentioned task, the present invention takes following technical solution:

A frozen semen dilution, characterized in that the prepared frozen semen dilution contains: sodium citrate: 0.9 grams to 1.2 grams, sucrose: 3 grams to 5 grams, egg yolk: 18 milliliters to 22 milliliters per 100 milliliters , Glycerin: 4ml-7ml, vitamin E: 6mg-9mg, vitamin C: 500mg-700mg, spectinomycin: 35mg, gentamicin 80,000 units (IU).

The preparation method of the above-mentioned frozen semen dilution is characterized in that it comprises the following steps:

Accurately weigh sodium citrate, sucrose, egg yolk, vitamin E, vitamin C and glycerin in the formula of claim 1, add distilled water to 100 ml, fully stir evenly on a magnetic stirrer to obtain a mixed solution, and then put it into a 4°C refrigerator overnight;

Before semen dilution, add spectinomycin milligram and gentamicin in the formula of claim 1 in mixed solution, and stir for 10 minutes;

Use acid or alkali to adjust the pH of the semen dilution between 6.7 and 7.2, and filter the dilution with a 0.22 micron filter to obtain a frozen semen dilution;

When in use, use a 0.22-micron filter to filter the frozen semen dilution, put it in a 37.5°C water bath for standby, and then follow the conventional semen production method to thaw the semen.

In the present invention, VE _{and VC} are combined into the semen diluent for the first time, and the preparation method of the diluent is revised. Experimental data show that the frozen semen diluent of the present invention not only improves the viability and The integrity rate of sperm acrosome, and the freezing of catalase (CAT), glutathione peroxidase (GR) and glutathione reductase (GSH) used to protect sperm fertilization ability in seminal plasma The vitality after thawing ensures that this semen formula can improve the quality of sperm after freezing.

Detailed ways

According to the technical scheme of the present invention, the substances needed to prepare 100 ml of frozen semen diluent are: sodium citrate: 0.9-1.2 grams, sucrose: 3-5 grams, egg yolk: 18-22 milliliters, glycerol: 4-7 milliliters, Vitamin E: 6-9 mg, vitamin C: 500-700 mg, spectinomycin: 35 mg, gentamicin: 80,000 units (IU).

Its best formula for frozen semen dilution is: the content of substances per 100ml is: sodium citrate: 1.073mg, sucrose: 4.344mg, egg yolk: 20ml, glycerin: 6ml, vitamin E: 8mg, vitamin C: 600 mg, spectinomycin: 35 mg, 80,000 units (IU) of gentamicin.

Its preparation method is:

1) Weigh the raw materials in the formula: sodium citrate, sucrose, egg yolk, glycerin, vitamin E, vitamin C, distilled water.

2) Prepare the above sodium citrate, sucrose, egg yolk, vitamin C, vitamin E, glycerin, and distilled water to form a base solution, stir with a magnetic stirrer for 60 minutes to obtain a mixed solution, and store it in a refrigerator at 4°C overnight for later use.

3) Before diluting the semen, add the formulated amount of gentamicin and spectinomycin to the mixed solution, and then stir for 10 minutes on a magnetic stirrer.

4) Adjust the pH value of the diluent to 6.5-7.5 with acid or alkali, and filter the diluent with a 0.22 micron filter to obtain a semen diluent.

The frozen semen diluent prepared in this example and the traditional formula were measured for bovine sperm viability, acrosome integrity and seminal plasma antioxidant enzymes after freezing. The seminal plasma antioxidant enzymes mainly included catalase (CAT), glutathione peroxidase (GR) and glutathione reductase (GSH).

The detection methods of sperm motility rate, acrosome integrity rate and seminal plasma antioxidant enzymes are as follows:

(1) Immediately after thawing the semen, check the viability at a temperature of 35°C to 37°C. When evaluating, use a glass rod to dip 1 drop of semen, drop it on a glass slide, add a cover glass, make the space between the cover glass and slide glass filled with semen, and then place it under a microscope at 400 to 600 times for observation. In the field of view of the microscope, the sperm motility is evaluated by comprehensive observation of the sperm activity in the upper, middle and lower layers of the semen.

(2) Take a drop of thawed semen and drop it on a clean glass slide, use another glass slide at 45 degrees, and smear the semen to the other side to make a smear. After natural air-drying, fix with 1-2 ml of formalin phosphate buffer for 15 minutes, wash and dry, and stain with Giemsa for 90 minutes, then remove the dye solution on the smear, and observe under a 1000-fold biological microscope after air-drying at 300 spermatozoa, the total number of spermatozoa and the number of intact acrosomes were recorded.

(3) Use relevant kits for measuring enzymes (the kits for measuring enzymes are all from Nanjing Jiancheng Biotechnology Co., Ltd.) to measure the activity of antioxidant enzymes in seminal plasma in strict accordance with the operating procedures.

The following is a specific test example given by the inventor. The test selects adult Holstein bulls with strong physique and strong libido, and raises them according to the breeding and management requirements of the bulls. The semen collection frequency is 3 times a week. Bull semen was collected by the false vagina collection method, and immediately after the semen collection, it was examined under a microscope at 35°C to 37°C. The collected semen is milky white or milky yellow, has no peculiar smell, the sperm morphology is normal, and the motility rate reaches above 0.7 before it can be used to make frozen semen.

Three different formulations of frozen semen dilutions and a traditional formulation were set up in this trial:

Formula 1: Prepare 100 ml of diluent: sodium citrate: 1.073 mg, sucrose: 4.344 mg, egg yolk: 20 ml, glycerin: 6 ml, vitamin E: 8 mg, vitamin C: 600 mg, spectinomycin: 35 mg, Gentamicin: 80,000 units (IU).

Recipe 2: Prepare 100 ml of diluent: sodium citrate: 1.073 mg, sucrose: 4.344 mg, egg yolk: 20 ml, glycerin: 6 ml, vitamin C: 600 mg, spectinomycin: 35 mg, gentamicin: 8 Thousands of units (IU).

Recipe 3: Prepare 100 ml of diluent: sodium citrate: 1.073 mg, sucrose: 4.344 mg, egg yolk: 20 ml, glycerin: 6 ml, vitamin E: 8 mg, spectinomycin: 35 mg, gentamicin: 8 Thousands of units (IU).

Traditional formula: prepare 100 ml of diluent: sodium citrate: 1.073 mg, sucrose: 4.344 mg, egg yolk: 20 ml, glycerin: 6 ml, spectinomycin: 35 mg, gentamicin: 80,000 units (IU).

Strictly weigh the ingredients according to the above formula: sodium citrate, sucrose, egg yolk, glycerin, vitamin E and vitamin C, and dilute to 100 ml with distilled water; stir on a magnetic stirrer for 60 minutes to fully stir to obtain a mixed solution, Then put it in a 4°C refrigerator overnight.

Before diluting the semen, add 35 mg of spectinomycin and 80,000 units (IU) of gentamicin to the mixed solution, and stir for 10 minutes. Adjust the pH of the diluent with acid or alkali, and measure the pH of the diluent at 6.7 with a pH meter. Between ~7.2, the semen dilution was obtained.

When in use, use a 0.22-micron filter to filter the diluted solution. After completion, place it in a water bath at 37.5°C. Then follow the conventional semen production method to thaw and dilute the frozen semen. After thawing, test the quality of the semen. See the specific indicators below. surface:

Effects of different formulations of frozen semen dilutions on the quality of frozen-thawed sperm (mean ± standard deviation)

deal with Recipe one Recipe 2 Recipe three   Traditional formula Live rate (%) 39.33±0.58a 37.33±0.58b 38.67±1.53ab 35.33±1.15c Complete rate of acrosome (%) 76.33±2.08a 74.33±1.53ac 75.67±1.53a 71.67±1.53bc Catalase activity (U/mL) 4.57±0.50a 3.99±0.12b 4.37±0.1ab 3.97±0.21b Glutathione peroxidase activity (U/L) 100.53±1.76a 48.48±2.93d 80.14±1.10b 74.76±1.56c Glutathione reductase activity (U/L) 136.67±2.06b 121.83±1.72c 165.98±1.73a 117.51±1.30c

Note: The different letters marked after the data of the same industry indicate significant differences (P<0.05), and the same letters marked indicate no significant differences (P<0.05).

As can be seen from the above table, the frozen semen diluent provided by the present invention has the highest performance in indicators such as sperm motility rate, acrosome integrity rate, catalase, glutathione peroxidase, and glutathione reductase activity. All were significantly better than other formulations (P<0.05). The formula one is respectively 5.6%-11.3% higher than the formula two or three in terms of sperm motility, and 11.3% higher than the traditional formula. In terms of acrosome integrity rate, formula 1 is about 3.7%-5.5% higher than other formulas and 6.5% higher than traditional formula. In terms of catalase activity, formula 1 is 0.5%-10.1% higher than other formulas, and 15.1% higher than traditional formula. The activity of glutathione peroxidase in formula 1 is 34.4% higher than that of traditional formula, and the activity of glutathione reductase is 16.31% higher than that of traditional common formula. Therefore, after the above analysis, it is concluded that the three formulas prepared by the frozen semen diluent prepared in this example can play the greatest role in protecting the quality of the sperm during the process of freezing and thawing sperm, and formula one is the best frozen semen diluent formula .

Claims (2)

1. a freezing seminal fluid dilution is characterized in that, contains in per 100 milliliters of this freezing seminal fluid dilution that makes: sodium citrate: 1.073 milligrams; Sucrose: 4.344 milligrams, yolk: 20 milliliters, glycerine: 6 milliliters; Vitamin E: 8 milligrams; Vitamin C: 600 milligrams, spectinomycin: 35 milligrams, gentamicin: 80,000 units.

2. the compound method of the described freezing seminal fluid dilution of claim 1 is characterized in that, comprises the following steps:

Accurately take by weighing sodium citrate, sucrose, yolk, vitamin E, vitamin C and glycerine in claim 1 prescription, adding distil water to 100 milliliter stirs on magnetic stirring apparatus, obtains mixed solution, puts into 4 ℃ of refrigerator overnight then;

Before the sperm dilution, in mixed solution, add the spectinomycin and the gentamicin of formula ratio, and stirred 10 minutes;

Between 6.7～7.2, dilution is carried out the bacteriological filtration processing with the acid-base value of acid or alkali adjustment semen diluent, obtain freezing seminal fluid dilution with 0.22 micron filter.