KR100903581B1 - Diluents for long-term preservation of swine liquid sperm - Google Patents

Abstract

The present invention relates to a long-term preservation diluent of pig liquid crystals used in artificial insemination, and more particularly to a long-term preservation diluent of pig liquid liquid containing a flavonoid agent having an antioxidant effect combined with one or more sugar (sugar). . The diluent of the present invention includes a flavonoid formulation having an antioxidant effect of inhibiting lipid metabolism of sperm cell membranes and inhibiting the process of pulverization after collection, and the survival rate and motility of sperm due to rancidity of sperm cell membrane lipids according to the preservation time. In addition, the sperm DNA damage, compared with the pH buffering agent and metabolic nutrient additives disclosed in the past, has an excellent effect of not affecting sperm motility, viability, and conception rate even when stored for 10 days or more.

Description

Diluent for long-term preservation of swine liquid sperm {Porcine Liquid Semen Diluent for Long Term Preservation}

The present invention relates to a long-term preservation diluent for liquid pig liquid sperm, the long-term preservation of liquid liquid sperm for maintaining the number of sperm, sperm appearance and vitality, etc. living in the sperm even if the long-term preservation of pig semen A diluent for solvents.

In recent years, along with the development of industry, genetic engineering has become an important discipline that can be used as a measure of the scientific development of the country, and this genetic engineering is the study necessary to preserve and develop high-quality varieties and further preserve human life. As it goes beyond the basic stage, it reaches a considerable level.

If natural breeding is carried out when breeding livestock or pets for human breeding, only one male and one female can be breeding, but if artificial insemination is made using liquid semen, Raw semen can be mixed with the diluent to increase the amount of semen and fertilize several females. Therefore, according to artificial insemination, semen having excellent genetic traits can be collected and fertilized by several females at the same time, so that it is possible to produce seeds of excellent lineage and preserve obedience.

The development of diluents is an important factor for the success of artificial insemination methods that have such advantages. Although research on this has been started for several decades, cryopreservation method of sperm that can be applied to various animals and livestock has been developed and used. In the case of sperm physiological characteristics, sperm cryopreserved due to low fertility rate is artificially inseminated using liquid semen, but the development of a diluent that can preserve the semen in liquid form for a long time is currently insufficient.

Domestic hog farmers have increased the size of pig breeding to full-time commercialization, and the form of breeding consists of collaborative or complexization, and the need for pig artificial insemination is increasing. Most pig farmers are carrying out artificial fertilization. However, the establishment of artificial insemination technology using pig liquid sperm is essential for the commercialization and expansion of artificial insemination of pigs.However, most of the diluents necessary for artificial insemination of pigs are imported from foreign countries, or they are produced domestically after receiving diluent manufacturing technology. Relying on the use of cloning, the diluent prepared in the laboratory by domestic researchers, egg yolk (egg yolk), pH preservatives, metabolic nutrients are added to the fact that the retention period is less than three to four days. Therefore, there has been a strong demand for the development of artificial insemination diluent for pig semen, which is not inferior to natural seed in terms of conception rate and delivery rate, and can be industrialized.

Lipids present in sperm cell membranes have been reported to oxidize over time and to drastically reduce motility, structural integrity and viability of sperm head acrosome (Michael R et al., 2004. Sperm chromatin structure assay (SCSA) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles.Fertility & Sterility.Vol 81. N0.5 1289-1295; Charlotte D. et al., 2004. Boar sperm storage capacity of BTS and androhep plus: viability, motility, capacitation, and tyrosine phosphosrylation.Theriogenology 62. 874--886). The oxidation of lipids is known to be due to the effects of free radicals that occur during metabolic processes, and because mammalian sperm, especially swine sperm, have high levels of unsaturated fatty acids and relatively few antioxidants, they are very sensitive to the harmful effects of reactive oxygen species. The oxidative action that occurs over time after reacting and injecting is greatly influenced by fertilization ability of sperm by viability of sperm, motility, and bacterial growth. In the body, the complex antioxidant system present in semen and intestines has the ability to mitigate free radicals that occur under normal conditions. However, if the sperm is severely damaged when the antioxidant function is insufficient or excessively generated in vitro, studies are being actively conducted to alleviate reactive oxygen species. A type of oxidation is a strong activity adversely affecting the sperm plasma membrane oxygen species, ^{_{superoxide anion (O 2 -),}} peroxyl radical (ROO -), hydroxyl radical (OH -), hydrogen peroxide (H 2 O 2) , etc. are known In addition, antioxidants that mitigate the adverse effects of reactive oxygen species are known as enzyme-based and non-enzymatic antioxidants. Superoxide dismutase (SOD), catalase, and Glutathione Peroxidase (GSHPx) are representative enzyme-based antioxidants, and vitamin E ( α-tocopherol, vitamin C (ascorbic acid), carotenoids (carotenoids), most of the ubiquinol / quinine (coenzyme Q), flavonoids (flavonoids) and the like are known as non-enzymatic antioxidants.

Diluent preservatives with pH buffering agents and metabolic nutrients are used for the preservation of swine sperm in Korea, but virtually all dilutions have to be used within 3 days after dilution due to the rapid decrease in motility and survival of sperm from 3-4 days after preservation. Therefore, the frequency of semen collection from the sows and the distribution of semen is a problem, and the diluent is almost entirely dependent on imports from foreign countries, and domestic development and dissemination of new sperm diluent is urgently needed in accordance with the physiological characteristics of swine sperm.

Overseas, diluent stocks such as Beltsville Thawing Solution (BTS) developed in the United States, Androhep developed in Germany, Ensure ensured in Spain, and Modena and Zorlesco have been widely used. Tris, Citrate, EDTA, HEPES, BSA, etc. are added to prevent metabolic control, pH change suppression, and sperm condensation, but the shelf life is longer than that of domestic products. When stored for more than one day, the conception rate is limited due to low fertility rate due to lower survival rate and motility, and it can be seen that there is a difference from the present invention in the fundamental oxidation inhibition and sperm properties according to the storage time.

As such, until now, artificial insemination for breeding pigs has not been used smoothly due to the physiological characteristics of swine sperm, and most of the liquid sperm is used. However, liquid sperm has a shorter shelf life than frozen semen. Due to its disadvantages, it is not easy to utilize the excellent semen and the economic loss due to the decrease in fertility rate due to sperm motility and bacterial infection according to the retention period.

Thus, the present inventors are trying to find a way to use the pig liquid sperm for a long time by increasing the shelf life without damaging the sperm motility or sperm tofu acrosome, etc. used in pig artificial insemination, two sugar ( Dilution of semen from pig males using buffer preservation solution containing flavonoid antioxidants extracted from plants with 3-glucoside 7-glucuronide, Structural Formula 1) improves the harmful environment of sperm injected in vitro. The present invention has been completed by confirming the extension of semen retention time by preserving viability and vitality.

It is an object of the present invention to provide a diluent which enables long term preservation of liquid sperm according to the specificity of swine sperm. If the diluent of the present invention is used, semen can be stored for 10 days or more, and dilution does not damage the conception rate even when using the stored semen.

In order to achieve the above object, the present invention provides a long-term preservation diluent for liquid pig liquid comprising a flavonoid agent having an antioxidant effect combined with one or more sugar (sugar).

Hereinafter, the present invention will be described in detail.

In the diluent of the present invention, the flavonoids are preferably two sugars combined, more preferably 3-glucoside-7-glucuronide is bonded as shown in the following structural formula (1). The flavonoid includes a 3-glucoside 7-O-glucuronide structure in which glucose and glucuronic acid are bonded to carbon 3 and carbon 7 as shown in the following structural formula.

(Formula 1)

In the diluent of the present invention, the flavonoids are preferably extracted from plants, and preferably contain 0.01 to 10 mg per 100 ml of distilled water. It is preferable that the diluent of the present invention treats the liquid pig liquid at 10 to 22 ℃.

 In addition, in the diluent of the present invention, the diluent is a base composed of glucose, Sodium citrate, Sodium bicarbonate, EDTA, Potassium chloride, Potassium phosphate, Citric acid, Bovine Serum Albumin (Fraction V), glycine, Tris, and Trehalose in addition to flavonoids It is preferable to include a solution, the base solution is 1 ~ 10 g of glucose per 100 ml of distilled water, 0.2 ~ 3 g Sodium citrate, 0.05 ~ 1 g Sodium bicarbonate, 0.05 ~ 2 g EDTA, 0.01 ~ 2 g Potassium chloride, Potassium More preferably, 0.01 to 0.5 g of phosphate, 0.005 to 0.1 g of citric acid, 5 to 100 mg of Bovine Serum Albumin (Fraction V), 0.5 to 3 g of glycine, 0.5 to 30 g of Tris and 0.01 to 1 g of Trehalose.

In addition, it is preferable that the basic solution further contains an antibiotic selected from the group consisting of gentamycin neomycin and apramycin as an antibiotic, and finally, the diluent is 100 ml of distilled water. Sugar flavonoids 0.01-10 mg, glucose 1-10 g, Sodium citrate 0.2-3 g, Sodium bicarbonate 0.05-1 g, EDTA 0.05-2 g, Potassium chloride 0.01-2 g, Potassium phosphate 0.01-0.5 g, Citric acid 0.005 ~ 0.1 g, Bovine Serum Albumin (Fraction V) 5-100 mg, glycine 0.5-3 g, Tris 0.5-30 g, Trehalose 0.01-1 g and gentamicin 50-10,000 IU are most preferred.

The long-term preservation diluent of pig liquid sperm of the present invention contains a flavonoid (structure 1), which is a non-enzymatic antioxidant extracted from a plant in a natural state, so that survival and motility of sperm due to rancidity of sperm cell membrane lipids according to semen preservation time, Sperm DNA damage is an excellent new diluent that does not affect sperm motility, viability or even conception even when stored for 10 days or more compared to the pH buffer and metabolic nutrient additives disclosed in the prior art.

Among the components used in the long-term preservation diluent of liquid pig liquids according to the present invention glucose is a metabolic substrate and energy source. Sodium citrate, Sodium bicarbonate, Bovine Serum Albumin, EDTA, Potassium chloride, Potassium phosphate, Citric acid, glycine, Tris and Trehalose are used to control sperm buffering, acidity (pH) and osmotic pressure, and to relieve temperature shock. to be. In addition, the antibiotics gentamycin (neomycin), neomycin (neomycin), or apramycin (apramycin) is to prevent the degradation of sperm viability due to bacterial contamination in liquid semen with prolonged shelf life. The diluent according to the present invention maintains the sperm viability and vitality of the pig semen can be stored for a long time to maintain the normal conception rate and delivery rate.

The process for preparing a long-term preservation diluent of liquid pig liquid of the present invention is as follows.

(1) Flavonoid Extraction

The leaves of the plants are harvested and lyophilized, extracted with ethanol, concentrated by filtration, concentrated with chloroform and ethyl acetate in a solvent, and concentrated first. The extract is recovered by each component by high performance liquid chromatography, and the components are obtained by using two-dimensional chromatography and ultraviolet spectroscopy to obtain glycosides in which one or more sugars, preferably two or more kinds of sugars, are combined (structure formula). One). After that, the organic solvent component is completely removed and freeze-dried and used as an antioxidant.

(2) Preparation and Selection of Antioxidant-Added Sperm Diluent

The basic composition of sperm diluent is prepared based on inorganic salts such as Glucose, Sodium citrate, Sodium bicabonate, Bovine Serum Albumin (Fraction V), EDTA, Potassium chloride, Potassium phosphate, Citric acid, glycine, Tris, Trehalose . The flavonoid glycoside compounds prepared by extracting the base solution are added at various concentrations, pHs, and antibiotics to prepare various combinations of solutions. After diluting the pig sperm using the solution to confirm the sperm motility according to the number of days of storage to first select a composition with excellent vitality.

(3) sperm test according to preservation time

Freshly collected semen is immediately subjected to computer assisted sperm analysis to select samples of individuals with good motility, and then diluted and stored in various diluents. Sperm viability over time using flow cytometer In addition, motility (mitochondrial function test) is performed and compared, and sperm chromatin structure assay is performed to compare with those on the market for the presence of harmful components.

The present invention provides a diluent comprising a flavonoid formulation having an antioxidant effect that inhibits lipid metabolism of sperm cell membranes and inhibits the process of being discarded after collection, thereby surviving sperm viability and motility due to rancidity of sperm cell membrane lipids over time. In addition, the sperm DNA damage, compared with the pH buffering agent and metabolic nutrient additives disclosed in the past, has an excellent effect of not affecting sperm motility, viability, and conception rate even when stored for 10 days or more. Therefore, it is possible to preserve the long-term storage of liquid liquid pigs difficult to store until now, it is possible to preserve the breed of pigs excellent in lineage, and to improve the fertility rate by artificial insemination of pig semen in pigs at the appropriate time and place.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples and may be changed to other embodiments equivalent to substitutions and equivalents without departing from the technical spirit of the present invention. Will be apparent to those of ordinary skill in the art.

Example 1 Antioxidant Flavonoid Extraction

The leaves of 10 plants belonging to hardwoods were harvested and lyophilized. This was continuously extracted and filtered for 3 days using 85% and 50% methanol, and concentrated through a reduced pressure evaporation method in a constant temperature water bath of 40 ° C. or lower using a rotary evaporator. Then, this was centrifuged at 3,000 rpm for 20 minutes using chloroform to extract only the pigment component from which chlorophyll was removed. The extract was removed with an anthocyanin component through a solvent fraction using ethyl acetate to separate only flavonoid components. Then, it was concentrated using a rotary evaporator to obtain a primary concentrated stock extract.

The primary concentrated stock extract was developed on high performance liquid chromatography using acetonitrile and 2% acetic acid as a mobile phase to recover each component, and concentrated again to separate flavonoid components purely. Each purely separated substance is identified by the two-dimensional chromatography, ultraviolet spectroscopy, and the like based on respective development rate values, emission time, and spectral data, and then glycosides in which two or more sugars of flavonoid components are combined 1) was separated. Then, the organic solvent component was completely removed using ethyl alcohol and distilled water, and only pure flavonoid crystals were obtained by freeze-drying and stored frozen and used as an antioxidant.

Example 2 Preparation of Diluent and Semen Dilution

The mixture having the following composition was uniformly mixed using a mixer to prepare a pig semen diluent.

0.1 mg of flavonoid glycoside (Formula 1) per 100 ml of distilled water, 4 g of glucose, 0.7 g of Sodium citrate, 0.12 g of Sodium bicabonate, 30 mg of Bovine Serum Albumin (Fraction V), 0.15 g of EDTA, 0.1 g of Potassium chloride, 0.05 g of Potassium phosphate Mix well to contain 0.02 g of Citric acid, 0.3 g of Glycine, 6 g of Tris, 0.1 g of Trehalose, and 900 IU of gentamycin.

The semen freshly collected by the penis hydraulic method using the diluent prepared above was diluted with 2 million sperm per 1 ml of diluent. At this time, the collection process of the semen is obvious to those skilled in the art, so the detailed description thereof will be omitted.

Test Example Effect of Liquid Slurry Diluent on Sperm Vitality

Freshly collected semen is immediately divided into three groups (Androhep (A), Beltsville Thowing Solution (B), and diluent Q of the present invention), diluted to 20 million sperm per ml of diluent, and stored in a semen reservoir at 17 ° C. According to the storage days, fluorescence staining was performed for sperm viability, motility (mitochondrial test), DNA damage (chromatin chromatin structure), and then the test was performed using a FACS Aria flow cytometer. At this time, the composition of Androhep (A) contains 0.26 g of glucose, 0.8 g of sodium citrate, 0.24 g of EDTA, 0.12 g of sodium bicarbonate, 0.9 g of HEPES, and 25 mg of Bovine Serum Albumin per 100 ml. In addition, the components of Beltsville Thowing Solution (B) contain 0.37 g of glucose, 0.6 g of sodium citrate, 0.125 g of EDTA, 0.125 g of sodium bicarbonate, and 75 mg of potassium chloride per 100 ml of distilled water. Each test is described in more detail below.

Test Example 1 Sperm Survival Test

Sperm cell viability test was performed by a basic observation on a 200 x microscope, followed by biostaining for more objective and accurate analysis, and analyzed by a flow cytometer (flowcytometer). Specifically, each group of diluted sperm cells were stored with the SYBR-14 fluorescent dye (the living cells emit green fluorescence by a blue laser light source (488 nm) and the fluorescence signal by the 530/30 nm band filter). Condensed} and propidium iodide fluorescent dye (dead cells emit red fluorescence by a blue laser light source (488 nm) and fluorescent signals are collected by a 610/20 nm band filter) at room temperature for 5 minutes. So. The survival rate of sperm by preservation time was analyzed by analyzing the ratio of green and red fluorescence in the average 20 million sperm transferred to the flow cytometer (Fig. 1).

As a result, there was no significant difference in the sperm survival rate in the three groups for the first four days after preservation, but from the 5th day after the dilution preservation, the A and B family began to degrade, especially in the B product. Noticeably. On the other hand, Q, a diluent of the present invention, was shown to maintain the initial survival rate relatively stably until 10 days after the preservation.

Test Example 2 Sperm Motility Test

For sperm vitality test, we investigated mitochondrial activity in sperm, which is the energy source of sperm. Specifically, Rhodamine-123 (a cell with normal mitochondrial function is treated with a blue laser light source (488 nm)) that can specifically stain the mitochondrial membrane for a more accurate and objective vitality test after observing basic vitality under a 200 x microscope. Fluorescence signal is condensed in the 575/26 ㎚ wavelength range) In order to select dead sperm together with fluorescent dye, propidium iodide fluorescent dyes are stained for 10 minutes at room temperature, respectively. Comparison was made by retention time using an analyzer (FIG. 2).

As a result, it was confirmed that sperm motility showed a similar pattern as in sperm viability test. Throughout the entire testing period, Q maintained no significant change in sperm motility, suggesting that there is no problem in conception rate even when artificial insemination using sperm preserved for more than 10 days.

Test Example 3 Sperm DNA Damage Test

Sperm DNA damage caused by oxidation of sperm membrane lipids over days of preservation was examined. Specifically, a normal double-stranded DNA strand is dyed with Acridine Orange fluorescent dye and emits green fluorescence by a blue laser light source (488 nm), whereby a fluorescent signal is collected by a 530/30 nm band filter. On the other hand, the damaged single-stranded DNA structure emits red fluorescence by a blue laser light source (488 nm), and the fluorescence signal is collected by a 610/20 nm band filter. The two fluorescent dyes listed above were stained at room temperature for 5 minutes, and the fluorescence ratios over time were compared and analyzed by flow cytometry in an average of 20,000 sperm (FIG. 3).

As a result, from the 7th day after preservation, damage to the double stranded DNA strands occurred in the A and B families, which reported a high correlation between the known changes in sperm chromatin structure and male infertility. (D'Occhio et al., 2007. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival.Anim Reprod Sci. 101 (1-2): 1-17) Implementing this implies that the conception rate may be lowered. In contrast, the Q group shows almost stable light emission, indicating that stable fertility can be maintained after a considerable period of time.

Test Example 4 Sperm Oxidation Test

In order to measure the degree of sperm oxidation according to the days of preservation, the pH change was measured by taking 5 ml of each semiprepared semen stored in each product, including an undiluted undiluted solution using a pH meter (Fig. 4). .

As a result, raw semen, which was not diluted in the preservative after collection, showed rapid oxidation after 4 days due to lipid metabolism as expected, and group A began to descend to acidity after 4 days, whereas group B Rather, the pH rises to alkalinity (temporary increase in metabolism, which results in a temporary increase in vitality, but ultimately in a short time). The excellence was confirmed.

1 is a graph showing the sperm survival rate when using the conventional diluent A and B family and the diluent Q of the present invention,

2 is a graph showing sperm motility in the case of using the conventional diluent A and B family and the diluent Q of the present invention,

3 is a graph showing the presence or absence of abnormality of sperm DNA double helix structure in the case of using the conventional diluent A and B family and the diluent Q of the present invention,

4 is a graph showing the pH change of semen when using the conventional diluent A and B family and the diluent Q of the present invention.

Claims (10)

Pigment liquid long-term preservation diluent comprising a flavonoid agent with an antioxidant effect, 3-glucoside-7-glucuronide is bonded as shown in the following structural formula 1. (Formula 1)

delete delete delete The long-term preservation diluent for liquid pig liquids according to claim 1, wherein the flavonoid contains 0.01 to 10 mg per 100 ml of distilled water. The method of claim 1, wherein the diluent comprises a basic solution consisting of glucose, Sodium citrate, Sodium bicarbonate, EDTA, Potassium chloride, Potassium phosphate, Citric acid, Bovine Serum Albumin (Fraction V), glycine, Tris, and Trehalose in addition to flavonoids. Pig liquid sperm long-term preservation diluent, characterized in that. The method of claim 6, wherein the base solution is 1 ~ 10 g of glucose per 100 ml of distilled water, 0.2 ~ 3 g Sodium citrate, 0.05 ~ 1 g Sodium bicarbonate, 0.05 ~ 2 g EDTA, 0.01 ~ 2 g Potassium chloride, Potassium phosphate 0.01 ~ 0.5 g, Citric acid 0.005 ~ 0.1 g, Bovine Serum Albumin (Fraction V) 5 ~ 100 mg, glycine 0.5 ~ 3 g, Tris 0.5 ~ 30 g and Trehalose 0.01 ~ 1 g Long-term preservative diluent. [7] The long-term preservation diluent of pig liquid tablets according to claim 6, wherein the basic solution further contains an antibiotic selected from the group consisting of gentamycin neomycin and apramycin as an antibiotic. . According to claim 5, wherein the diluent is 0.01 to 10 mg of flavonoids per 100 ml of distilled water, glucose 1 ~ 10 g, Sodium citrate 0.2 ~ 3 g, Sodium bicarbonate 0.05 ~ 1 g, EDTA 0.05 ~ 2 g, Potassium chloride 0.01 ~ 2 g, Potassium phosphate 0.01-0.5 g, Citric acid 0.005-0.1 g, Bovine Serum Albumin (Fraction V) 5-100 mg, glycine 0.5-3 g, Tris 0.5-30 g, Trehalose 0.01-1 g and gentamicin 50- Long term liquid preservative diluent for pig liquids comprising 10,000 IU. The method of claim 1, wherein the diluent pig liquid sperm long-term preservation diluent, characterized in that for treating pig liquid sperm at 10 to 22 ℃.

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