CN105994251B - A kind of dilution preservation liquid for killing animal semen virus and bacteria - Google Patents

Abstract

The present invention provides a kind of dilutions for killing animal semen virus and bacteria to preserve liquid, by weight, including following component：0 0.002 parts of 25 40 parts of glucose, 2.5 3 parts of citric acid, 58 parts of trisodium citrate, 1.2 1.3 parts of sodium bicarbonate, 1.2 1.3 parts of disodium ethylene diamine tetraacetate, 0.7 0.9 parts of potassium chloride, 2.8 3 parts of trishydroxymethylaminomethane, 0.05 0.08 parts of cysteine, 0.025 0.030 parts of povidone iodine and antibacterial peptide.For the dilution of economic animal sperm, preservation and kill bacterial virus.

Description

A dilute preservation solution for killing animal semen virus bacteria

technical field

The invention belongs to the field of artificial insemination of animals in animal husbandry, and in particular relates to a diluted preservation solution for killing virus and bacteria in animal semen.

Background technique

The advantage of artificial insemination technology is that it can collect high-quality animal semen manually, and through certain technical means, it can be stored and transported for a long time, which will bring huge economic benefits to the improvement of animal husbandry varieties and production performance. However, artificial insemination technology also has its natural defect, that is, it can infect all pathogenic microorganisms that may be contained in the donor's semen to its recipient. Taking pigs as an example, boar semen usually contains 10 ⁴ ~10 ⁵ CFU/ml of bacteria and possibly contaminated unknown viruses and other pathogenic microorganisms. These bacteria and viruses will affect the storage time of boar semen and the viability of sperm, and can transmit pathogenic microorganisms that boars may carry to sows and piglets through artificial insemination techniques, resulting in reproductive disorders in sows and piglet diseases. The epidemic and the sharp drop in production performance caused serious economic losses.

Studies by domestic and foreign scholars have shown that the main viruses that can be detected in boar semen and endanger pig production are: foot-and-mouth disease virus (FMDV), swine fever virus (CSFV), porcine circovirus (PCV-2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), Japanese Japanese encephalitis virus (JEV). Among them, it has been confirmed that CSFV, PRRSV, PPV, and PRV can infect sows through artificial insemination and cause reproductive disorders such as stillbirth and abortion. Semen infection of boars is an important way for the vertical transmission of viral diseases in swine herds, and it is also a major problem restricting the development and promotion of artificial insemination technology.

Animal semen dilution preservation solution is widely used in the artificial insemination industry. Its technical difficulty is not high, and its formula is simple and easy to produce. survival time. However, for the contamination of boar semen, the existing technology is to add antibiotics (such as penicillin, streptomycin, gentamicin, lincomycin, etc.) Some of the bacteria in the body multiply, but they are completely ineffective against viruses, and there is a risk of overuse of antibiotics. Therefore, there is currently no better way to deal with the bacterial and viral contamination in semen in the field of artificial insemination of economical animals.

For example, the invention patent application 201510756292.6 discloses a long-acting diluted pig fresh essence preservation powder and its preparation method and application. It is composed of glucose 35-40 in parts by weight, sodium oxalate tetraacetate 1.2-1.4, sodium citrate 4-8, sodium bicarbonate 1.2-1.4, potassium chloride 0.7-0.8, polyvinyl alcohol 0.4- 0.6, cysteine 0.1-0.2, inositol 0.4-0.6, penicillin 1.0-1.5, streptomycin 1.0-1.5 and other raw materials, there is a problem of abuse of antibiotics.

Invention patent application 201410164618.1 discloses the composition of a porcine antiviral semen dilution. Based on per 1000 ml of double distilled water, porcine antiviral semen dilution contains: 15.50 parts of glucose; 11.65 parts of trisodium citrate; 2.35 parts of EDTA sodium salt; 1.75 parts of sodium bicarbonate; 1.00 parts of polyvinyl alcohol; 5.50 parts 4.10 parts of citric acid; 0.07 parts of cysteine; 1.00 parts of vitamin C; 0.10 parts of enrofloxacin; and 50ml of antiviral agent. The formula contains antiviral ingredients such as ribavirin. Although ribavirin has a certain effect of inhibiting the growth of viruses, it is an antiviral drug that is strictly prohibited by the country for animal husbandry and veterinary industries.

The difficulty of developing the semen dilution preservation solution for killing animal semen virus bacteria is reflected in:

a. Safety, first of all, it must not damage the sperm viability, not affect the reproduction of fertilized eggs, embryos and sows, and have no effect on the genetic properties of sperm.

b. Effectiveness, effective against harmful pathogens in sperm, especially viruses, is the key to this technology.

c. It can provide effective protection for sperm, and the storage period of sperm can reach 7-10 days.

d. Safety and effectiveness are a pair of contradictions, and it is difficult to unite opposites.

e. High requirements for research conditions, such as pharmacy, veterinary bacteriology, virology, virus cell culture, and animal husbandry and other related majors and experimental conditions, are difficult for a single enterprise or research unit to meet.

The literature "The effect of a semen purification and protection agent on the viability of boar sperm and the killing effect on 5 viruses in semen" (Chinese Journal of Animal Husbandry, Reproductive Physiology, 2015, Vol. 51, No. 7), published a The effect of a semen purification and protection agent on the motility of boar sperm, and its killing effect on viruses present in boar semen. The semen purification and protection agent of this document is not suitable for iodine-containing disinfectants.

Contents of the invention

In order to solve the above technical problems, the present invention provides a diluted preservation solution for killing virus and bacteria in animal semen. Adding low toxicity povidone-iodine and antibacterial peptides into the boar semen dilution and preservation solution is used for diluting, preserving and killing bacteria and viruses in the semen of economical animals.

In order to realize the above-mentioned purpose of the invention, the present invention adopts the following technical solutions:

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 25-40 parts of glucose, 2.5-3 parts of citric acid, 5-8 parts of trisodium citrate, and 1.2-1.3 parts of sodium bicarbonate , 1.2-1.3 parts of disodium edetate, 0.7-0.9 parts of potassium chloride, 2.8-3 parts of trishydroxymethylaminomethane, 0.05-0.08 parts of cysteine, 0.025-0.030 parts of povidone-iodine and 0-0.002 parts of antimicrobial peptides.

Preferably, by weight, the following ingredients are included: 35-40 parts of glucose, 2.8-3 parts of citric acid, 5-6 parts of trisodium citrate, 1.25-1.3 parts of sodium bicarbonate, 1.25- 1.3 parts, 0.7-0.8 parts of potassium chloride, 2.8-2.9 parts of tris, 0.05-0.07 parts of cysteine, 0.025-0.030 parts of povidone-iodine and 0.001-0.002 parts of antimicrobial peptides.

This diluted preservation solution is divided into two parts, the first part is the basic diluted nutrient solution: glucose, citric acid, trisodium citrate, sodium bicarbonate, disodium edetate, potassium chloride, trishydroxymethylaminomethane and cysteine, wherein cysteine is a sperm protection agent; the second part is the effective ingredient for killing viruses and bacteria: povidone-iodine. The formula of the present invention controls the addition ratio of trishydroxymethylaminomethane, edetate disodium and potassium chloride, so that the pH value of the diluted preservation solution is 6.20-6.30, and the osmotic pressure is 347mOsm.

The pH of the diluted preservation solution of the present invention is 6.20-6.30, and the osmotic pressure is 347mOsm. For the strict environmental requirements of sperm survival, the storage stability of sperm is best in this environment.

The solvent used in the diluted preservation solution of the present invention is double distilled water.

Water quality has a great influence on the vitality and storage time of sperm. The water quality of different breeding enterprises in different regions is different. Key indicators such as pH, mineral and trace element content, microbial content, and heavy metal content have a great impact on sperm quality. The invention adopts stable double distilled water quality, and overcomes the disadvantages of inconvenient use and unstable effect.

Povidone-iodine is one of the iodine preparations commonly used in livestock and poultry farms. It is a contact disinfectant formed by loading iodine on a strong basic anion resin, which can kill most bacteria, fungi and viruses. However, within the safe concentration range for sperm, povidone-iodine has a good effect on some bacteria and viruses, but it cannot completely kill the five common viruses in semen, especially porcine pseudorabies virus and swine fever virus bad.

Antimicrobial peptide is a kind of biologically active small molecule polypeptide induced in organisms, with a molecular weight of about 2000-7000 and composed of 20-60 amino acid residues. It has a broad-spectrum killing effect on bacteria and viruses, and has a The safety of sperm is very good, especially it has a good killing effect on common viruses in semen, especially swine fever virus, pseudorabies virus, and PRRS virus, and it has a strong complementarity with povidone-iodine.

Further preferably, the antimicrobial peptide is an endogenous antimicrobial peptide of cockroaches.

The present invention selects cockroach endogenous antibacterial peptide, and its amino acid sequence is SEQ ID NO: 1. This antimicrobial peptide has the characteristics of good stability, broad-spectrum and high-efficiency antibacterial and antiviral properties, and the production process is mature and perfect. After the amino acid sequence is recombined, it is expressed and fermented with engineering bacteria to obtain pure recombinant broad-spectrum antimicrobial peptides. Cockroach endogenous antimicrobial peptides are very safe to sperm at an appropriate concentration, and have a good killing effect on common viruses in semen, especially swine fever virus, pseudorabies virus, and PRRS virus, and low-toxic polydimensional The combination of ketone-iodine has strong complementarity.

The diluted preservation solution of the present invention has been verified by more than 100 large and medium-sized pig raising enterprises across the country. After using the product of the present invention, the production performance has been greatly improved by more than 80%, which generally reflects that the reproductive disorders of sows are greatly reduced, and the incidence of diseases Significantly decreased, the pregnancy rate increased significantly, the number of litter piglets increased, and the production performance generally improved, with significant economic and social benefits.

The present invention adopts the preferred basic formula of diluted preservation solution and povidone-iodine disinfectant, so that the stability between the diluted preservation solution formulas is the best, coupled with uniform and stable double-distilled water quality, it overcomes the inconvenience of use and poor effect Stable disadvantages.

Detailed ways

The substantive content of the present invention will be further described in detail below in conjunction with specific embodiments.

Example 1

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 25 parts of glucose, 2.5 parts of citric acid, 5 parts of trisodium citrate, 1.2 parts of sodium bicarbonate, disodium edetate 1.2 parts, 0.7 parts of potassium chloride, 2.8 parts of tris, 0.05 parts of cysteine and 0.025 parts of povidone-iodine.

Example 2

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 40 parts of glucose, 3 parts of citric acid, 8 parts of trisodium citrate, 1.3 parts of sodium bicarbonate, disodium edetate 1.3 parts, 0.9 parts of potassium chloride, 3 parts of tris, 0.08 parts of cysteine, 0.030 parts of povidone-iodine and 0.002 parts of antimicrobial peptide.

The pH value of the diluted preservation solution is 6.2, and the osmotic pressure is 347 mOsm.

The solvent of the diluted preservation solution is double distilled water.

Example 3

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 30 parts of glucose, 2.6 parts of citric acid, 6 parts of trisodium citrate, 1.22 parts of sodium bicarbonate, disodium edetate 1.22 parts, 0.8 parts of potassium chloride, 2.9 parts of tris, 0.06 parts of cysteine and 0.026 parts of povidone-iodine.

The pH value of the diluted preservation solution is 6.3, and the osmotic pressure is 347 mOsm.

The solvent of the diluted preservation solution is double distilled water.

Example 4

A diluted preservation solution for killing animal semen virus bacteria, comprising the following components by weight: 35 parts of glucose, 2.8 parts of citric acid, 5 parts of trisodium citrate, 1.25 parts of sodium bicarbonate, disodium edetate 1.25 parts, 0.7 parts of potassium chloride, 2.8 parts of tris, 0.05 parts of cysteine, 0.025 parts of povidone-iodine and 0.001 parts of antimicrobial peptides.

The pH value of the diluted preservation solution is 6.22, and the osmotic pressure is 347 mOsm.

The solvent of the diluted preservation solution is double distilled water.

Example 5

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 40 parts of glucose, 3 parts of citric acid, 6 parts of trisodium citrate, 1.3 parts of sodium bicarbonate, disodium edetate 1.3 parts, 0.8 parts of potassium chloride, 2.9 parts of tris, 0.07 parts of cysteine, 0.030 parts of povidone-iodine and 0.002 parts of cockroach endogenous antimicrobial peptide.

The pH value of the diluted preservation solution is 6.25, and the osmotic pressure is 347 mOsm.

The solvent of the diluted preservation solution is double distilled water.

Example 6

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 37 parts of glucose, 2.9 parts of citric acid, 6 parts of trisodium citrate, 1.25 parts of sodium bicarbonate, disodium edetate 1.25 parts, 0.75 parts of potassium chloride, 2.8 parts of tris, 0.07 parts of cysteine, 0.026 parts of povidone-iodine and 0.0015 parts of cockroach endogenous antimicrobial peptides.

The pH value of the diluted preservation solution is 6.26, and the osmotic pressure is 347 mOsm.

The solvent of the diluted preservation solution is double distilled water.

Example 7

A diluted preservation solution for killing animal semen viruses and bacteria, comprising the following components by weight: 37 parts of glucose, 2.9 parts of citric acid, 6 parts of trisodium citrate, 1.25 parts of sodium bicarbonate, disodium edetate 1.25 parts, 0.75 parts of potassium chloride, 2.8 parts of tris, 0.08 parts of cysteine, 1 part of vitamin C, 0.026 parts of povidone-iodine and 0.0012 parts of cockroach endogenous antimicrobial peptides.

The pH value of the diluted preservation solution is 6.22, and the osmotic pressure is 347mOsm.

The solvent of the diluted preservation solution is double distilled water.

Toxicology test

Toxicology tests use animal experiments to observe the results of acute toxicity, subacute toxicity and irritant toxicity.

Results: The acute oral LD50 of the semen purification and disinfectant for rats and mice was greater than 5000 m parts/k parts·BW, and its stock solution was non-irritating to rabbit eyes, intact skin and damaged skin; the dosage was 1000 m parts Below /k part·BW, compared with the negative control group, it has no significant effect on the body weight, blood routine and blood biochemical indicators of the rats, and no obvious pathological changes are seen in the organs of the experimental animals. The piglets took orally the stock solution of the semen purification and protection agent twice a day, 1ml/time, for two consecutive days. Drug period. The toxicology of the semen purification and protection agent is safe at the concentration used.

The semen purifying disinfectant of the invention has good stability, and the iodine content decline rate is lower than 5% after being placed in a 37°C thermostat for three months, and the iodine content decline rate is lower than 10% after six months.

The present invention is used for the normal-temperature dilution preservation solution of porcine semen, which has good stability and can be stored in an airtight normal temperature and protected from light for a period of validity of up to one year. The pH is 6.20±0.05, and the osmotic pressure is 347mOsm.

Preparation method: In a sterile factory building, add the formulated amount of glucose, EDTA-Na2, trisodium citrate:, citric acid, sodium bicarbonate, Tris, potassium chloride and cysteine in 995.00ml sterile double distilled water at 37ºC in sequence acid, stir at low speed while adding, keep the temperature at 37 ºC, stir evenly and wait for the temperature to drop to room temperature, add povidone-iodine in the prescribed amount, stir evenly at low speed, then aseptically dispense.

Finished product inspection item and operation method of the present invention

1. Detection of available iodine content

According to the detection method of available iodine in "Veterinary Pharmacopoeia of the People's Republic of China" (2005 edition), the povidone iodine contained in this product is calculated as available iodine (I), which should be 0.85% to 1.20% of the labeled amount.

【Properties】This product is a colorless liquid.

【identification】

(1) Take 1-5 drops of this product, add 10ml of water and 1 drop of starch indicator solution, and it will appear blue-purple.

(2) Take 10ml of this product, put it in a 50ml Erlenmeyer flask (do not stain the inner neck of the bottle), cover the bottle mouth with a piece of filter paper moistened with starch indicator solution, leave it for 80 seconds, and no blue color will appear.

【Check】The pH value should be 6.20±0.05.

2. The influence of different brands of semen dilution preservation solution and semen dilution preservation powder on the storage time of semen at room temperature

Four different brands of semen dilution and preservation solutions or semen dilution and preservation powder (No. 1, No. 2, No. 3 and No. 4) are purchased in the market and diluted for later use.

Table 1 Effects of different dilutions on storage of porcine semen at room temperature

It can be seen from Table 1 that the effective storage time of the diluted preservation solution of the present invention is significantly higher than that of No. 1, 2, 3 and 4 solutions (P<0.01), and the survival index is also significantly higher than that of No. 1, 2, 3 and 4 solutions ( P<0.05).

3. Preservation results of semen breeding at room temperature in the diluted preservation solution of the present invention

The pig semen is diluted with the diluted preservation solution of the present invention, stored in a thermostat at 17°C for 5 days, and the diluted semen with a sperm motility above 0.5 is used for artificial insemination experiments. A total of 120 sows were inseminated, and the specific results are shown in Table 2: It can be seen that the conception rate and average litter size of pig semen stored in the diluted preservation solution of the present invention at room temperature for 5 days are significantly higher than those of No. 1, No. 2, No. 3 and No. 4 solutions.

Table 2 Conception rate and average litter size of pig semen stored at room temperature for 5 days in different dilutions

sequence listing

<110> Chengdu Meiqiang Veterinary Technology Service Co., Ltd.

<120> A Dilute Preservation Solution for Killing Animal Semen Viruses and Bacteria

<160>1

<170>PatentInversion3.3

<210>1

<211>37

<212>PRT

<213> Cockroach Endogenous Antimicrobial Peptides

<400>1

Lys Trp Lys Val Phe Lys Lys Ile glu Lys Met gly Arg Asn Ile Arg

1 5 10 15

Asn gly Ile Val Lys Ala gly Pro Ala Ile Ala Val Leu gly glu Ala

20 25 30

Lys Ala Lys Ala Leu

35

Claims (3)

1. a kind of dilution preservation liquid of killing porcine semen virus bacterium is characterized in that: by weight, is made up of following composition: 35-40 parts of glucose, 2.8-3 parts of citric acid, 5-6 parts of trisodium citrate, 1.25-1.3 parts of sodium bicarbonate, 1.25-1.3 parts of disodium edetate, 0.7-0.8 parts of potassium chloride, 2.8-2.9 parts of trishydroxymethylaminomethane, 0.05-0.07 parts of cysteine, polydimensional Ketone iodine 0.025-0.030 parts and antimicrobial peptide 0.001-0.002 parts; The pH value of the diluted preservation solution is 6.2-6.3, and the osmotic pressure is 347mOsm. 2. a kind of dilute storage solution for killing porcine semen virus bacteria according to claim 1, is characterized in that: the antimicrobial peptide of described dilute storage solution is cockroach endogenous antimicrobial peptide. 3. a kind of dilute preservation liquid that kills porcine semen virus bacteria according to claim 1, is characterized in that: the solvent of described dilute preservation liquid is double distilled water.