WO2010005260A2 - Diluent for long-term preservation of liquid boar sperm - Google Patents

Diluent for long-term preservation of liquid boar sperm Download PDF

Info

Publication number
WO2010005260A2
WO2010005260A2 PCT/KR2009/003788 KR2009003788W WO2010005260A2 WO 2010005260 A2 WO2010005260 A2 WO 2010005260A2 KR 2009003788 W KR2009003788 W KR 2009003788W WO 2010005260 A2 WO2010005260 A2 WO 2010005260A2
Authority
WO
WIPO (PCT)
Prior art keywords
sperm
diluent
liquid
long
pig
Prior art date
Application number
PCT/KR2009/003788
Other languages
French (fr)
Korean (ko)
Other versions
WO2010005260A3 (en
Inventor
손중호
황재홍
고정문
Original Assignee
주식회사 노아바이오텍
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 노아바이오텍 filed Critical 주식회사 노아바이오텍
Publication of WO2010005260A2 publication Critical patent/WO2010005260A2/en
Publication of WO2010005260A3 publication Critical patent/WO2010005260A3/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts

Definitions

  • 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.
  • diluents are an important factor for the success of artificial insemination methods that have such advantages.
  • cryopreservation method of sperm that can be applied to various animals and livestock has been developed and used.
  • 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.
  • Lipid oxidation is known to be due to the effects of free radicals that occur during metabolic processes, and because mammalian sperm, especially swine sperm, have a lot 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 by reacting and injecting is greatly influenced by fertilization ability of sperm by viability, motility and bacterial growth.
  • the complex antioxidant system present in semen and intestines has the ability to mitigate free radicals that occur under normal conditions.
  • the sperm is severely damaged when the antioxidant function is insufficient or excessively generated in vitro, recent 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
  • 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 ( ⁇ -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.
  • 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 it is urgent to develop and disseminate new sperm diluent in accordance with the physiological characteristics of swine sperm.
  • diluent preservation solutions such as Beltsville Thawing Solution (BTS) developed in the US, Androhep developed in Germany, Ensure ensured in Spain, and Modena, Zorlesco are used.
  • BTS Beltsville Thawing Solution
  • 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.
  • 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.
  • 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 artificial pig insemination, two sugars (3 Dilution of semen from pig males using buffer preservation solution containing quercetin antioxidant, a flavonoid extracted from a plant with -glucoside 7-glucuronide, Structural Formula 1), improved the harmful environment of sperm injected in vitro.
  • the present invention was completed by confirming that the semen preservation time was extended by preserving the viability and vitality.
  • the present invention provides a long-term preservation diluent of pig liquid liquid containing a quercetin glycoside preparation which is a kind of flavonoids having an antioxidant effect combined with two sugars (sugar).
  • the quercetin glycoside is preferably 3-glucoside-7-glucuronide bonded as shown in the following Structural Formula 1.
  • the flavonoids include 3-glucoside 7-O-glucuronide structure in which glucose and glucuronic acid are bonded to carbon 3 and carbon 7 as shown in Structural Formula 1 below.
  • the quercetin glycoside is preferably extracted from plants, and preferably contains 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 °C.
  • the diluent is 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 quercetin glycosides
  • the basic solution is 1 to 10 g of glucose per 100 ml of distilled water, 0.2 to 3 g of sodium citrate, 0.05 to 1 g of sodium bicarbonate, 0.05 to 2 g of EDTA, 0.01 to 2 g of Potassium chloride, More preferably, 0.01 to 0.5 g of Potassium 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 .
  • the basic solution is more preferably contained antibiotics selected from the group consisting of gentamycin neomycin (neomycin), and apramycin (antimycin) as an antibiotic, after all, the diluent per 100 ml of distilled water Quercetin glycoside 0.01 to 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.
  • antibiotics selected from the group consisting of gentamycin neomycin (neomycin), and apramycin (antimycin) as an antibiotic, after all, the diluent per
  • Long-term preservation diluent of pig liquid sperm of the present invention contains a quercetin glycoside (structure 1), which is a non-enzymatic antioxidant flavonoid extracted from plants in a natural state, the survival rate of sperm due to the rancidity of sperm cell membrane lipids over time semen preservation And 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 buffering agent and metabolic nutrient additives disclosed above in motility and sperm DNA damage.
  • structure 1 is a non-enzymatic antioxidant flavonoid extracted from plants in a natural state
  • 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.
  • 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.
  • flavonoids are currently classified and identified, all of which belong to polyphenols.
  • the main component of the conventionally used polyphenol is catechin derived from green tea (catechin), whereas the present invention provides a quercetin glycoside, which is a flavonoid in which two sugars are bound.
  • Flavonoids exist in a variety of biosynthetic processes, where catechin is a polyphenol having the structure of 'flavan-3-ol', whereas the quercetin, the flavonoid specified in the present invention, has a completely different structure with the structure of 'flavonol'. Polyphenols.
  • flavonoid biosynthesis pathway is described as follows. Flavonoids of various structures are present by various enzymes using phenylalanine as a precursor. Catechins are synthesized by passing 3-OH-flavanones directly to flavan instead of flavonol, the process by which quercetin glycosides are made, while quercetin glycosides are flavonoids derived from flavonols. The chemical reaction of the biosynthesis process representing this is shown in FIG. 5.
  • Flavonoids described in the prior art, etc. are mainly derived from catechins in a sugar-unbound form, whereas the present invention uses synthetic quercetin glycosides, ie, flavonoids in which two sugars are bound, as described above. Is completely different and its functions are also different.
  • catechins are relatively biosynthetic, whereas the quercetin glycosides used in the present invention are very difficult to synthesize in general because it is not easy to bind the desired sugar to the desired site in each ring structure.
  • Flavonoids present in plants in nature are all commonly known to be sugar-bonded state, the quercetin glycosides used in the present application is very difficult to synthesize in general because it binds the desired sugar to the desired site in each ring structure Because it is not easy to let.
  • flavonoids described in the present application are not flavonoids commonly present in all plants.
  • the present invention extracts the quercetin glycoside, which is a 3-glucoside-7-glucuronide-bound flavonoid, and demonstrates their effects, the other two sugar-bound flavonoids, quercetin glycoside, show similar functions. I think that.
  • the present invention significantly improved the preservation of liquid sperm in the liquid state by using a specific quercetin glycoside to inhibit or delay the oxidation process of the sperm of the pig.
  • the antioxidant used in the present invention utilizes a quercetin glycoside in which two sugars are bound, and the present invention provides the sperm physiological characteristics of porcine sperm cells. Due to the high lipid content, cryopreservation is difficult and the emphasis is on the prevention of rancidity due to the preservation time in the liquid state.
  • the meaning of the sugar-bound quercetin is solubility that the sugar-containing flavonoids are easily dissolved in water, but the sugar-bonded flavonoids can be used only when dissolved in organic solvents such as DMSO or ethanol, and dissolved in organic solvents.
  • the quercetin used in the present invention is an antioxidant that has an antioxidant effect similar to that of catechin, which is present in a water-soluble state (not in a crystalline state) outside the cell in the antioxidant effect and passes through the cell membrane while the sugar falls through the cell membrane. It is an antioxidant that is markedly different from.
  • the process for preparing a long-term preservation diluent of liquid pig liquid of the present invention is as follows.
  • Flavonoid Extracts Quercetin Glycosides
  • 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 quercetin glycosides in which one or more of the flavonoid components, preferably two or more kinds of sugars are bound ( Structural Formula 1). After that, the organic solvent component is completely removed and freeze-dried and used as an antioxidant.
  • 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 prepared flavonoid glycoside compounds extracted by the base solution are added at various concentrations, pHs, and antibiotics to prepare various combination 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.
  • Freshly collected semen is immediately subjected to computer assisted sperm analysis to select samples of individuals with good motility, diluted in various diluents, and stored in various diluents.
  • Sperm viability over time using flowcytometer 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 quercetin glycoside preparation, which is an flavonoid with an antioxidant effect that inhibits lipid metabolism of sperm cell membranes and inhibits the process of post-harvesting, thereby preventing sperm due to rancidity of sperm cell membrane lipids over time. Survival, motility, and sperm DNA damage, compared with the pH buffer and metabolic nutrient additives disclosed in the past 10 days or more, there is an excellent effect that does not affect the motility, viability, and even conception rate of sperm.
  • 1 is a graph showing sperm viability when using the conventional diluent A and B family and the diluent Q of the present invention
  • FIG. 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
  • Figure 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
  • FIG. 5 is a schematic diagram showing the synthesis of catechin and quercetin glycosides using phenylalanine as a precursor for flavonoid biosynthesis, and is a reference diagram to show that catechin and quercetin are different.
  • 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 quercetin glycosides having two or more sugars of the flavonoid component bound together ( Structural Formula 1) was isolated. 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.
  • the mixture having the following composition was uniformly mixed using a mixer to prepare a pig semen diluent.
  • flavonoid quercetin glycoside (Structure 1) per 100 ml of distilled water, glucose 4 g, 0.7 g Sodium citrate, 0.12 g Sodium bicabonate, 30 mg Bovine Serum Albumin (Fraction V), 0.15 g EDTA, 0.1 g Potassium chloride, Potassium phosphate 0.05 g, Citric acid 0.02 g, glycine 0.3 g, Tris 6 g, Trehalose 0.1 g, and gentamicin 900 IU were mixed well.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 dyes (the dead cells emit red fluorescence by a blue laser light source (488 nm) and a fluorescent signal is collected by a 610/20 nm band filter) for 5 minutes at room temperature. . 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).
  • Rho-damine-123 the cells with normal mitochondrial function are treated with a blue laser light source (488 nm), which can specifically stain mitochondrial membranes for more accurate and objective vitality testing after observing basic vitality under a 200 x microscope.
  • a blue laser light source (488 nm)
  • propidium iodide fluorescent dyes were stained for 10 minutes at room temperature to average 20,000 sperm in sperm was compared by preservation time using a flow cytometer (Fig. 2).
  • 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.
  • 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).
  • 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). .

Abstract

The present invention relates to a diluent for long-term preservation of liquid boar sperm used in artificial fertilization. More particularly, the invention relates to a diluent for long-term preservation of liquid boar sperm including a quercertin glycoside agent, a type of flavonoid having antioxidant effect which has a dual sugar linkage. The diluent of this invention has excellent effect and does not affect sperm motility or conception rate compared to previously reported pH buffer agents and metabolic nutritional supplements in terms of sperm survival rate, motility, and sperm DNA damage due to rancidity of sperm cell membrane lipids over time even if sperm is preserved for over 10 days because it includes a quercertin agent of the flavonoid having antioxidatant effect, which inhibits the rancidity process after extraction by inhibiting the lipid metabolism of sperm cell membranes.

Description

돼지 액상정액 장기보존을 위한 희석제Diluents for long-term preservation of swine liquid sperm
본 발명은 돼지 액상정액 장기보존 희석제에 관한 것으로, 돼지 정액을 장기 보존한 경우에도 정액 내에 살아 있는 정자의 수, 정자의 성상과 활력 등이 10일 이상 그대로 유지될 수 있게 하는 돼지 액상 정액 장기보존용 희석제에 관한 것이다.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.
근래 산업의 발달과 더불어 유전공학은 그 국가의 과학발전을 가늠할 수 있는 척도라 할 만큼 중요한 학문으로 자리 잡고 있으며, 이러한 유전공학은 우량 품종을 보존 개발하고, 나아가 인류의 생명을 보존하기 위해 필요한 학문으로서 기초 단계를 넘어 상당한 수준에 이르고 있다.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.
인간이 사육하고 있는 가축이나 애완동물의 번식을 위하여 종부를 시킬 때에 자연종부를 실시하게 되면 수컷 1마리와 암컷 1마리밖에 종부시킬 수 없으나, 액상 정액을 이용하여 인공수정을 시키면 한번 채취한 수컷의 원 정액을 희석액과 혼합하여 정액량을 증가시켜 여러 마리의 암컷에 수정을 시킬 수 있다. 따라서 인공수정에 의하면 우수한 유전형질을 가진 정액을 채취하여 동시에 여러 마리의 암컷에 수정이 가능하므로 혈통이 우수한 종자의 생산 및 순종의 보존이 가능해진다.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.
이러한 장점을 가지고 있는 인공수정 방법이 성공하기 위해서는 희석제의 개발이 중요한 관건이 되는데, 이에 대한 연구가 수십 년 전부터 시작되어 여러 동물 및 가축에 적용할 수 있는 정자의 동결 보존법은 개발되어 사용되고 있으나, 돼지의 경우 정자 생리학적 특성상 동결 보존된 정자로는 수태율이 너무 저조하여 액상 정액을 이용하여 인공수정을 하고 있는 실정이나, 정액을 액상형상으로 장기 보존할 수 있는 희석제에 대한 개발은 현재 미비한 시점이다.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.
국내의 양돈 농가들은 양돈사육 규모가 전업화로 확대되고 사육 형태가 협업 또는 단지화로 구성되면서 돼지 인공수정에 대한 필요성이 높아지고 있으며 대부분의 양돈농가들이 인공수정을 실시하고 있다. 그러나 돼지의 인공수정 실용화와 확대보급을 위해서는 돼지 액상정액을 이용한 인공수정기술 확립이 급선무이지만, 돼지의 인공수정에 필요한 희석제는 대부분 외국산을 도입하여 이용하고 있거나 희석제 제조기술을 전수 받아서 국내에서 제조하는 복제사용에 의존하고 있는 실정이며, 국내의 연구진에 의해 실험실에서 제조된 희석제는 계란의 노른자(난황), pH 보존제, 대사성 영양소가 첨가된 정도이며 이 역시 보존기간이 3~4일 미만인 실정이다. 따라서, 수태율 및 분만율 등의 측면에서 자연 종부와 비교하여 열등하지 않으며 산업화가 가능한 돼지 정액의 인공수정용 희석제의 개발이 강력하게 요구되어 왔다.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 which can be industrialized.
정자 세포막에 존재하는 지질은 시간이 지남에 따라 산화가 발생하고 정자의 운동성, 정자 두부 첨체의 구조적 온전성 및 생존성을 급격히 감소시키는 것으로 보고되어 있다(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). 지질의 산화는 대사 과정 중에 발생하는 활성산소계의 영향으로 알려져 있으며, 포유류의 정자 특히 돼지 정자는 불포화 지방산을 많이 가지고 있는 반면 상대적으로 적은 항산화제를 가지고 있기 때문에 활성산소종의 유해한 영향에 매우 민감하게 반응하고 사출되어 시간이 경과함에 따라 발생하는 산화작용로 인하여 정자의 생존성, 운동성저하, 세균증식 등에 의해 수정 능력에 큰 영향을 받게 된다. 체내에서는 정액과 정장 내에 존재하는 복합 항산화제 계통은 정상적인 상태 하에서 발생하는 활성산소종을 완화할 수 있는 기능이 있다. 그러나 체외조건에서 항산화제 기능이 부족하거나 과도하게 발생될 경우 정자는 심각한 손상을 받게 되므로 최근에는 활성 산소종을 완화시킬 수 있는 연구가 활발히 진행되고 있다. 정자 세포막에 악영향을 미치는 산화 작용이 강력한 활성 산소종의 종류로는 superoxide anion(O2 -), peroxyl radical(ROO-), hydroxyl radical(OH-), hydrogen peroxide(H2O2) 등이 알려져 있으며, 이와 같은 활성 산소종의 악영향을 완화시키는 항산화제로는 효소계 항산화제와 비효소계 항산화제가 알려져 있는데, Superoxide dismutase(SOD), catalase, Glutathione Peroxidase(GSHPx) 등이 대표적인 효소계 항산화제이며, 비타민E(α-tocopherol), 비타민C(ascorbic acid), 카로티노이드(carotenoids) 거의 대부분, ubiquinol/quinine(coenzyme Q), 플라보노이드(flavonoids) 등이 비효소계 항산화제로 알려져 있다.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 acrosomes (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., Boar sperm storage capacity of BTS and androhep plus: viability, motility, capacitation, and tyrosine phosphosrylation.Theriogenology 62. 874--886). Lipid oxidation is known to be due to the effects of free radicals that occur during metabolic processes, and because mammalian sperm, especially swine sperm, have a lot 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 by reacting and injecting is greatly influenced by fertilization ability of sperm by viability, 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, recent 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.
국내에서는 돼지정액의 보존을 위하여 pH 완충작용제 및 대사성 영양소 첨가 희석 보존액이 사용되고 있으나, 사실상 거의 모든 희석액이 보존 후 3~4일부터는 정자의 운동성 및 생존율이 급격히 저하되어 희석 후 3일 이내에 사용하여야 되기 때문에 종모돈으로부터의 정액채취빈도와 정액의 유통상에 문제가 되고 있어 희석제는 거의 전량 외국으로부터 수입에 의존하고 있고 돼지 정자의 생리 특성에 맞추어 새로운 정자 희석액의 국내 개발과 보급이 시급한 실정이다.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. As a result, 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 it is urgent to develop and disseminate new sperm diluent in accordance with the physiological characteristics of swine sperm.
국외의 경우 미국에서 개발한 Beltsville Thawing Solution(BTS) 이나, 독일에서 개발된 Androhep, 그리고 스페인에서 개발된 Ensure와 그 외에도, Modena, Zorlesco 라는 희석 보존액이 보급되어 사용되어 지고 있으며 이들 제품에는 버퍼용액 조성에 Tris, Citrate, EDTA, HEPES, BSA 등을 첨가하여 대사조절, pH 변화억제, 정자의 응결 등을 방지하는 수준으로 국내 제품에 비해서는 보존기간이 다소 길다고는 하나, 이들 제품 역시 3~7일 이상 보존했을 때 생존율 및 운동성 저하로 인하여 수태율이 저조하기 때문에 사용시간에 제약이 있고, 보존 시간경과에 따른 근본적인 산화 억제와 정자 성상에 있어 본 발명과는 상이한 차이가 있다고 볼 수 있다.Overseas, diluent preservation solutions such as Beltsville Thawing Solution (BTS) developed in the US, Androhep developed in Germany, Ensure ensured in Spain, and Modena, Zorlesco are 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.
현재까지 돼지의 번식을 위한 인공수정에는 돼지 정자의 생리학적 특성상 동결정액이 원활하게 사용되지 못하고 있고 거의 대부분 액상정액이 사용되어지고 있으나, 액상정액은 동결보존된 정액에 비해 보존기간이 짧은 단점이 있어 우수한 웅돈의 정액 활용이 원활하지 못하고 보존기간에 따른 정자 운동성 저하 및 세균감염등으로 인한 수태율 저하로 인한 엄청난 경제적인 손실을 가져오고 있는 실정이다. So far, artificial crystals for the breeding of pigs have not been used smoothly due to the physiological characteristics of pig sperm and almost all liquid sperm are used. However, liquid sperm has a shorter shelf life than frozen semen. The use of semen is not easy to use, and the situation is causing a huge economic loss due to a decrease in fertility rate due to a decrease in sperm motility and bacterial infection according to the preservation period.
이에, 본 발명자들은 돼지 인공수정에 사용되는 돼지 액상정액을 정자 운동성이나 정자 두부 첨체 등에 손상되지 않으면서도 보존기간을 늘려 돼지 액상정액을 장기간 사용할 수 있는 방법을 찾고자 노력하던 중, 두 개의 당(3-glucoside 7-glucuronide, 구조식 1)이 붙어있는 식물에서 추출한 플라보노이드인 쿼세틴 항산화제가 함유된 버퍼 보존액을 이용하여 돼지수컷에서 채취한 정액을 희석한 결과, 체외로 사출된 정자가 처하는 여러 유해 환경을 개선해 주어 생존성과 활력을 보존시킴으로써 정액보존 시간을 연장시켜 줌을 확인함으로써 본 발명을 완성하였다.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 artificial pig insemination, two sugars (3 Dilution of semen from pig males using buffer preservation solution containing quercetin antioxidant, a flavonoid extracted from a plant with -glucoside 7-glucuronide, Structural Formula 1), improved the harmful environment of sperm injected in vitro. The present invention was completed by confirming that the semen preservation time was extended by preserving the viability and vitality.
본 발명의 목적은 돼지 정자 특이성에 따른 액상정액의 장기보존을 가능하게 하는 희석제를 제공하는 것이다. 상기 본 발명의 희석제를 이용하면 정액을 10일 이상 보관이 가능하고, 희석하여 상기 보관된 정액을 이용하여도 수태율에 전혀 손상을 주지 않는다.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.
상기 목적을 달성하기 위하여, 본 발명은 2개의 당(sugar)이 결합된 항산화 효과가 있는 플라보노이드류(flavonoids)의 일종인 쿼세틴(quercetin) 배당체 제재를 포함하는 돼지 액상정액 장기보존 희석제를 제공한다.In order to achieve the above object, the present invention provides a long-term preservation diluent of pig liquid liquid containing a quercetin glycoside preparation which is a kind of flavonoids having an antioxidant effect combined with two sugars (sugar).
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 희석제에 있어서, 상기 쿼세틴 배당체는 하기 구조식 1과 같이 3-glucoside-7-glucuronide가 결합된 것이 바람직하다. 상기 플라보노이드는 하기 구조식 1과 같이, 3번 탄소와 7번 탄소위치에 포도당과 글루쿠론산이 결합된 3-glucoside 7-O-glucuronide 구조를 포함하고 있다.In the diluent of the present invention, the quercetin glycoside is preferably 3-glucoside-7-glucuronide bonded as shown in the following Structural Formula 1. The flavonoids include 3-glucoside 7-O-glucuronide structure in which glucose and glucuronic acid are bonded to carbon 3 and carbon 7 as shown in Structural Formula 1 below.
(구조식 1)(Formula 1)
Figure PCTKR2009003788-appb-I000001
Figure PCTKR2009003788-appb-I000001
또한, 본 발명의 희석제에 있어서, 상기 쿼세틴 배당체는 식물로부터 추출되는 것이 바람직하고, 증류수 100 ㎖ 당 0.01 내지 10 ㎎을 함유하는 것이 바람직하다. 본 발명의 상기 희석제는 10 내지 22℃에서 돼지 액상정액을 처리하는 것이 바람직하다.In the diluent of the present invention, the quercetin glycoside is preferably extracted from plants, and preferably contains 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 ℃.
또한, 본 발명의 희석제에 있어서, 상기 희석제는 쿼세틴 배당체 이외에 포도당, Sodium citrate, Sodium bicarbonate, EDTA, Potassium chloride, Potassium phosphate, Citric acid, Bovine Serum Albumin(Fraction V), glycine, Tris, 및 Trehalose로 구성된 기본용액을 포함하는 것이 바람직하고, 상기 기본용액은 증류수 100 ㎖ 당 포도당 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 ㎎, glycine 0.5~3 g, Tris 0.5~30 g 및 Trehalose 0.01~1 g를 포함하는 것이 보다 바람직하다. 아울러, 상기 기본용액은 추가적으로 항생제로 겐타마이신(gentamycin) 네오마이신(neomycin), 및 아프라마이신(apramycin)으로 구성된 군중에서 선택된 항생제를 함유하는 것이 보다 바람직하고, 결국, 상기 희석제는 증류수 100 ㎖ 당 쿼세틴 배당체 0.01 내지 10 ㎎, 포도당 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 ㎎, glycine 0.5~3 g, Tris 0.5~30 g, Trehalose 0.01~1 g 및 겐타마이신 50~10,000 IU를 포함하는 것이 가장 바람직하다.In addition, in the diluent of the present invention, the diluent is 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 quercetin glycosides It is preferable to include a basic solution, the basic solution is 1 to 10 g of glucose per 100 ml of distilled water, 0.2 to 3 g of sodium citrate, 0.05 to 1 g of sodium bicarbonate, 0.05 to 2 g of EDTA, 0.01 to 2 g of Potassium chloride, More preferably, 0.01 to 0.5 g of Potassium 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, the basic solution is more preferably contained antibiotics selected from the group consisting of gentamycin neomycin (neomycin), and apramycin (antimycin) as an antibiotic, after all, the diluent per 100 ml of distilled water Quercetin glycoside 0.01 to 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.
본 발명의 돼지 액상정액 장기보존 희석제는 천연물 상태로 식물에서 추출한 비효소계 항산화물질인 플라보노이드인 쿼세틴 배당체(구조식 1)를 함유하고 있어서, 정액보존 시간경과에 따른 정자 세포막 지질의 산패로 인한 정자의 생존율 및 운동성, 정자 DNA 손상에 있어 종래에 개시된 pH 완충작용제 및 대사성 영양소 첨가제에 비해 10일 이상 보존하여도 정자의 운동성이나, 생존성, 나아가 수태율에 영향을 끼치지 않는 탁월한 새로운 희석제이다.Long-term preservation diluent of pig liquid sperm of the present invention contains a quercetin glycoside (structure 1), which is a non-enzymatic antioxidant flavonoid extracted from plants in a natural state, the survival rate of sperm due to the rancidity of sperm cell membrane lipids over time semen preservation And 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 buffering agent and metabolic nutrient additives disclosed above in motility and sperm DNA damage.
본 발명에 따른 돼지 액상정액의 장기보존용 희석제에 사용되는 성분 중 포도당은 대사기질 및 에너지원으로 작용한다. Sodium citrate, Sodium bicarbonate, Bovine Serum Albumin, EDTA, Potassium chloride, Potassium phosphate, Citric acid, glycine, Tris 및 Trehalose 등은 정자의 완충작용, 및 산도(pH)와 삼투압을 조절하고, 온도 충격을 완화하기 위함이다. 아울러, 항생제인 겐타마이신(gentamycin), 네오마이신(neomycin), 혹은 아프라마이신(apramycin)은 보존기간의 연장에 따른 액상 정액 내의 세균오염에 의한 정자의 생존성 저하를 방지하기 위함이다. 본 발명에 따른 희석제는 돼지 정액의 정자 생존성 및 활력을 유지시켜 정액을 장기간 보존할 수 있으며 정상적인 수태율과 분만율을 유지하게 해준다.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.
플라보노이드(flavonoids)는 현재 4,000여종 이상이 분류 및 확인되어지고 있으며, 이들은 모두 폴리페놀에 속하는 물질이다. 일반적으로, 종래에 사용된 폴리페놀의 주성분은 녹차에서 유래된 카테킨(catechin)이 주성분인데 반하여, 본원발명은 2개의 당이 결합된 플라보노이드인 퀘세틴(quercetin) 배당체를 제공한다. 플라보노이드는 다양한 생합성 과정이 존재하고 있는데, 카데킨은 '플라반-3-올'의 구조를 갖는 폴리페놀인 반면에, 본 발명에 명시된 플라보노이드인 쿼세틴은 '플라보놀'의 구조를 갖는 전혀 다른 구조의 폴리페놀이다.More than 4,000 flavonoids are currently classified and identified, all of which belong to polyphenols. In general, the main component of the conventionally used polyphenol is catechin derived from green tea (catechin), whereas the present invention provides a quercetin glycoside, which is a flavonoid in which two sugars are bound. Flavonoids exist in a variety of biosynthetic processes, where catechin is a polyphenol having the structure of 'flavan-3-ol', whereas the quercetin, the flavonoid specified in the present invention, has a completely different structure with the structure of 'flavonol'. Polyphenols.
참고적으로, 플라보노이드 생합성 경로를 설명하면 다음과 같다. 페닐알라닌을 전구체로 하여 다양한 효소에 의하여 다양한 구조의 플라보노이드가 존재하게 된다. 카테킨은 3-OH-flavanones가 쿼세틴 배당체가 만들어지는 과정인 플라보놀로 가지 않고 바로 플라반으로 넘어가서 합성되는 반면에, 쿼세틴 배당체는 플라보놀로부터 유래된 플라보노이드이다. 이를 나타내는 생합성 과정의 화학 반응을 도 5에 나타내었다.For reference, the flavonoid biosynthesis pathway is described as follows. Flavonoids of various structures are present by various enzymes using phenylalanine as a precursor. Catechins are synthesized by passing 3-OH-flavanones directly to flavan instead of flavonol, the process by which quercetin glycosides are made, while quercetin glycosides are flavonoids derived from flavonols. The chemical reaction of the biosynthesis process representing this is shown in FIG. 5.
종래 문헌 등에 기재된 플라보노이드는 주로 당이 결합되지 않은 형태인 카테킨류에서 유래하는 반면에, 본원발명은 당이 결합된 쿼세틴 배당체, 즉 2개의 당이 결합된 플라보노이드를 이용하는 것으로 상기에서 지적한 바와 같이 합성경로가 완전히 다르며 그 기능도 다르다. 특히, 카테킨은 비교적 생합성이 용이한데 반하여, 본 발명에 사용된 쿼세틴 배당체는 일반적인 합성이 매우 어려운데 그 이유로는 각각의 링 구조에 원하는 부위에 원하는 당을 결합시키는 것이 쉽기가 않기 때문이다.Flavonoids described in the prior art, etc. are mainly derived from catechins in a sugar-unbound form, whereas the present invention uses synthetic quercetin glycosides, ie, flavonoids in which two sugars are bound, as described above. Is completely different and its functions are also different. In particular, catechins are relatively biosynthetic, whereas the quercetin glycosides used in the present invention are very difficult to synthesize in general because it is not easy to bind the desired sugar to the desired site in each ring structure.
자연계에서 식물에 존재하는 플라보노이드류는 모두 당이 결합된 상태인 것으로 보편적으로 알려져 있으며, 본 출원에 사용된 쿼세틴 배당체는 일반적인 합성이 매우 어려운데 그 이유로는 각각의 링 구조에 원하는 부위에 원하는 당을 결합시키는 것이 쉽기가 않기 때문입니다. 또한 식물마다 다양한 종류 및 함량의 플라보노이드가 존재하고 있으며, 본 출원에 명시된 플라보노이드는 모든 식물에 공통적으로 존재하고 있는 플라보노이드는 아니다. 비록 본원발명에서는 실시예에서 3-glucoside-7-glucuronide가 결합된 플라보노이드인 쿼세틴 배당체를 추출하고, 이들의 효과를 입증하고 있지만, 기타 다른 2개의 당이 결합된 플라보노이드인 쿼세틴 배당체도 유사한 기능을 나타내리라 판단된다.Flavonoids present in plants in nature are all commonly known to be sugar-bonded state, the quercetin glycosides used in the present application is very difficult to synthesize in general because it binds the desired sugar to the desired site in each ring structure Because it is not easy to let. In addition, there are various kinds and contents of flavonoids in each plant, and the flavonoids described in the present application are not flavonoids commonly present in all plants. Although the present invention extracts the quercetin glycoside, which is a 3-glucoside-7-glucuronide-bound flavonoid, and demonstrates their effects, the other two sugar-bound flavonoids, quercetin glycoside, show similar functions. I think that.
또한, 돼지 정자의 경우 다른 동물에 비해 지질함유가 너무 높아 동결보존이 안되고 또한 시간 경과에 따른 지질대사의 진행으로 산패하기 쉽다. 그 결과, 돼지 정자의 경우 다른 동물과 달리 동결보존이 불가능하고 액상으로 보존해야 한다. 따라서, 본원발명은 돼지 정자의 산화과정을 억제내지 지연시키기 위해 특정 쿼세틴 배당체를 이용하여 액상상태의 돼지정자의 보존을 현저하게 향상시켰다.In addition, in the case of swine sperm compared to other animals, the lipid content is too high, cryopreservation is not easy, and also prone to rancidity due to the progress of lipid metabolism over time. As a result, unlike other animals, pig sperm cannot be cryopreserved and must be preserved in liquid form. Therefore, the present invention significantly improved the preservation of liquid sperm in the liquid state by using a specific quercetin glycoside to inhibit or delay the oxidation process of the sperm of the pig.
지구상에는 아주 다양한 항산화제가 존재하고 있으나, 페닐기를 바탕으로 한 플라보노이드 중에 본 발명에 사용된 항산화제는 두 개의 당이 결합된 쿼세틴 배당체를 이용하는 것이며, 본원발명은 돼지정자세포가 가지는 정자생리학적 특징인 높은 지질함유로 인해 동결보존이 어려우며 액상상태로 보존 시간경과에 따른 산패방지효과에 중점을 둔 것이다. 당이 결합된 쿼세틴이 갖는 의미는 용해성에 있어서 당이 함유된 플라보노이드는 물에 용이하게 용해가 되나, 당이 결합되지 않은 플라보노이드는 DMSO나, 에탄올 등 유기용매에 녹여야 사용가능하며, 유기용매에 용해하였더라도 다른 혼합물에 첨가하여 사용하는 경우 다시 결정화되어 산화효과가 제한적일 수밖에 없다. 또한 본 발명에 사용된 쿼세틴은 항산화 효과에 있어 세포 바깥에서 (결정상태가 아닌) 수용성 상태로 존재하다가 세포막을 통과할 때는 당이 떨어져 나가면서 세포막을 통과하는 구조로 항산화 효과가 카테킨과 유사한 항산화제와는 현저하게 차별화되는 항산화제이다.Although there are a great variety of antioxidants on the planet, among the phenyl-based flavonoids, the antioxidant used in the present invention utilizes a quercetin glycoside in which two sugars are bound, and the present invention provides the sperm physiological characteristics of porcine sperm cells. Due to the high lipid content, cryopreservation is difficult and the emphasis is on the prevention of rancidity due to the preservation time in the liquid state. The meaning of the sugar-bound quercetin is solubility that the sugar-containing flavonoids are easily dissolved in water, but the sugar-bonded flavonoids can be used only when dissolved in organic solvents such as DMSO or ethanol, and dissolved in organic solvents. Even if it is used in addition to other mixtures, it is crystallized again and the oxidation effect is inevitably limited. In addition, the quercetin used in the present invention is an antioxidant that has an antioxidant effect similar to that of catechin, which is present in a water-soluble state (not in a crystalline state) outside the cell in the antioxidant effect and passes through the cell membrane while the sugar falls through the cell membrane. It is an antioxidant that is markedly different from.
본 발명의 돼지 액상정액의 장기보존용 희석제의 제조과정은 다음과 같다.The process for preparing a long-term preservation diluent of liquid pig liquid of the present invention is as follows.
(1) 플라보노이드(쿼세틴 배당체) 추출(1) Flavonoid Extracts (Quercetin Glycosides)
식물의 잎을 수확 및 동결건조 하여 메탄올 등을 이용하여 추출 및 여과 농축하고, 클로로포름 및 에틸아세테이트로 용매 등으로 농축시켜 1차 농축한다. 상기 추출물을 고성능 액체크로마토그래피로 각 성분별로 회수하고, 상기 성분들을 2차원 크로마토그래피 및 자외선 분광기 등을 이용하여 플라보노이드 성분 중 하나 이상, 바람직하게는 두 종류 이상의 당이 결합된 쿼세틴 배당체를 얻어낸다(구조식 1). 이후 유기용매 성분을 완전히 제거하고 동결 건조를 통하여 냉동 보관하며 항산화제로 사용한다.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 quercetin glycosides in which one or more of the flavonoid components, preferably two or more kinds of sugars are bound ( Structural Formula 1). After that, the organic solvent component is completely removed and freeze-dried and used as an antioxidant.
(2) 항산화제 첨가된 정자 희석액의 제조 및 선발(2) Preparation and Selection of Antioxidant-Added Sperm Diluent
정자 희석액의 기본 조성으로는 Glucose, Sodium citrate, Sodium bicabonate, Bovine Serum Albumin (Fraction V), EDTA, Potassium chloride, Potassium phosphate, Citric acid, glycine, Tris, Trehalose 등 무기 염류를 기반으로 기본 용액을 제조한다. 이 기본 용액에 추출하여 상기 준비된 플라보노이드 배당체 화합물들을 다양한 농도 및 pH, 항생제 별로 첨가하여 여러 조합의 용액을 제조한다. 상기 용액을 이용하여 돼지 정자를 희석하여 보존 일수에 따른 정자 운동성을 우선적으로 확인한 후 활력이 우수한 조성을 선발한다.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 prepared flavonoid glycoside compounds extracted by the base solution are added at various concentrations, pHs, and antibiotics to prepare various combination 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) 보존 시간경과에 따른 정자 성상 검사 (3) sperm test according to preservation time
신선하게 채취된 정액은 즉시 정자 운동성 검사(computer assisted sperm analysis)를 실시하여 운동성이 양호한 개체의 것을 시료로 선택한 후 여러 희석제에 희석 보존한 후 유세포 분석기(flowcytometer)를 이용하여 시간경과에 따른 정자 생존율, 운동성(미토콘드리아 기능검사)을 실시하여 비교하고, 아울러 정자 DNA 구조 이상 유무(sperm chromatin structure assay)를 실시하여 위해 성분이 있는지에 대해 시판되고 있는 제품들과 비교한다.Freshly collected semen is immediately subjected to computer assisted sperm analysis to select samples of individuals with good motility, diluted in various diluents, and stored in various diluents. Sperm viability over time using flowcytometer 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.
본 발명은 정자 세포막의 지질 대사를 억제하여 채취 후 산폐되는 과정을 억제하는 항산화 효과가 있는 플라보노이드인 쿼세틴 배당체 제재를 포함하는 희석제를 제공함으로써, 보존 시간경과에 따른 정자 세포막 지질의 산패로 인한 정자의 생존율 및 운동성, 정자 DNA 손상에 있어 종래에 개시된 pH 완충작용제 및 대사성 영양소 첨가제에 비해 10일 이상 보존하여도 정자의 운동성이나, 생존성, 나아가 수태율에 영향을 끼치지 않는 탁월한 효과가 있다. 따라서, 지금까지 보관이 곤란한 돼지 액상정액을 장기보존할 수 있어서 혈통이 우수한 돼지의 품종을 보존할 수 있고, 돼지의 정액을 적절한 시기 및 장소에서 암돼지에 인공수정하여 수태율을 높일 수 있다.The present invention provides a diluent comprising a quercetin glycoside preparation, which is an flavonoid with an antioxidant effect that inhibits lipid metabolism of sperm cell membranes and inhibits the process of post-harvesting, thereby preventing sperm due to rancidity of sperm cell membrane lipids over time. Survival, motility, and sperm DNA damage, compared with the pH buffer and metabolic nutrient additives disclosed in the past 10 days or more, there is an excellent effect that does not affect the motility, viability, and even conception rate of sperm. Therefore, it is possible to preserve the long-term preservation of liquid liquid pigs difficult to store until now, it is possible to preserve the breed of pigs excellent in bloodline, and to improve the fertility rate by artificial insemination of pig semen in pigs at the appropriate time and place.
도 1은 종래의 희석제 A와 B 제품군 및 본 발명의 희석제 Q를 사용한 경우의 정자 생존율을 나타내는 그래프이고, 1 is a graph showing sperm viability when using the conventional diluent A and B family and the diluent Q of the present invention,
도 2는 종래의 희석제 A와 B 제품군 및 본 발명의 희석제 Q를 사용한 경우의 정자 운동성을 나타낸 그래프이고, 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,
도 3은 종래의 희석제 A와 B 제품군 및 본 발명의 희석제 Q를 사용한 경우의 정자 DNA 2중 나선 구조의 이상 유무를 퍼센트로 나타낸 그래프이고, 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,
도 4는 종래의 희석제 A와 B 제품군 및 본 발명의 희석제 Q를 사용한 경우의 정액의 pH 변화를 나타낸 그래프이고, Figure 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,
도 5는 플라보노이드 생합성 경로로서, 페닐알라닌을 전구체로 하여 카테킨 및 쿼세틴 배당체의 합성 과정을 나타낸 도식도로서, 카테킨과 쿼세틴이 다른 것임을 보여주기 위한 참고적 도면이다. FIG. 5 is a schematic diagram showing the synthesis of catechin and quercetin glycosides using phenylalanine as a precursor for flavonoid biosynthesis, and is a reference diagram to show that catechin and quercetin are different.
이하, 본 발명을 하기 실시예에 의거하여 보다 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐, 본 발명은 하기 실시예에 의해 한정되는 것이 아니고, 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 치환 및 균등한 타 실시예로 변경할 수 있음은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 명백할 것이다.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.
<실시예 1 > 항산화제 플라보노이드 추출Example 1 Antioxidant Flavonoid Extraction
활엽수에 속하는 식물 10 종의 잎을 수확하여 동결 건조시켰다. 이것을 85% 및 50% 메탄올을 이용하여 3일간 연속 추출 및 여과하고 회전증발기를 이용하여 40℃ 이하의 항온 수조에서 감압증발 방법으로 통하여 농축하였다. 그리고 나서, 이를 클로로포름을 이용하여 3,000 rpm의 원심력으로 20분간 원심분리하여 엽록소를 제거한 색소성분만을 추출하였다. 상기 추출물을 에틸아세테이트를 이용하여 용매 분획을 통하여 안토시아닌 성분을 제거하여 플라보노이드 성분들만을 분리해내었다. 그리고 나서, 이를 회전 증발기를 이용하여 농축시켜 1차 농축 원액 추출물을 확보하였다.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.
상기 1차 농축 원액 추출물을 아세토나이트릴과 2% 초산을 이동상으로 하여 고성능 액체크로마토그래피에 전개시켜 각 성분별로 회수하고 이를 다시 농축하여 플라보노이드 성분들을 순수 분리하였다. 순수 분리된 각 물질들은 2차원 크로마토그래피 및 자외선 분광기 등을 이용하여 각각의 전개율 값 및 방출 시간 그리고 스펙트럼 자료에 의거하여 성분을 동정한 후, 플라보노이드 성분 중 두 종류 이상의 당이 결합된 쿼세틴 배당체(구조식 1)를 분리하였다. 그리고 나서, 에틸알콜과 증류수를 이용하여 유기용매 성분을 완전히 제거하고 동결 건조를 통하여 순수한 플라보노이드 결정들만을 얻어내어 냉동 보관하며 항산화제로 사용하였다.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 quercetin glycosides having two or more sugars of the flavonoid component bound together ( Structural Formula 1) was isolated. 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.
<실시예 2 > 희석제의 제조 및 정액 희석Example 2 Preparation of Diluent and Semen Dilution
다음과 같은 조성을 갖는 혼합물을 혼합기를 이용하여 균일하게 혼합하여 돼지 정액 희석제를 제조하였다.The mixture having the following composition was uniformly mixed using a mixer to prepare a pig semen diluent.
증류수 100 ㎖ 당 플라보노이드 쿼세틴 배당체(구조식 1) 0.1 ㎎, 포도당 4 g, Sodium citrate 0.7 g, Sodium bicabonate 0.12 g, Bovine Serum Albumin(Fraction V) 30 ㎎, EDTA 0.15 g, Potassium chloride 0.1 g, Potassium phosphate 0.05 g, Citric acid 0.02 g, glycine 0.3 g, Tris 6 g, Trehalose 0.1 g, 및 겐타마이신 900 IU를 함유하도록 잘 섞어주었다.0.1 mg of flavonoid quercetin glycoside (Structure 1) per 100 ml of distilled water, glucose 4 g, 0.7 g Sodium citrate, 0.12 g Sodium bicabonate, 30 mg Bovine Serum Albumin (Fraction V), 0.15 g EDTA, 0.1 g Potassium chloride, Potassium phosphate 0.05 g, Citric acid 0.02 g, glycine 0.3 g, Tris 6 g, Trehalose 0.1 g, and gentamicin 900 IU were mixed well.
상기 제조한 희석제를 이용하여 신선하게 음경 수압법으로 채취된 정액을, 희석액 1 ㎖당 200 만개의 정자로 희석시켰다. 이때, 상기 정액의 채취 과정은 당업자에게 명백한 사항이라 자세한 내용은 생략한다.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.
<시험예> 액상정액 희석제가 정자활력에 미치는 영향 테스트Test Example Effect of Liquid Slurry Diluent on Sperm Vitality
신선하게 채취된 정액을 즉시 3개의 그룹으로 나뉘어(Androhep (A), Beltsville Thowing Solution (B), 그리고 본 발명의 희석제 Q), 희석액 1 ㎖당 2000만 마리의 정자로 희석하여 17℃의 정액 보관고에 보관하면서 보관 경과일수에 따라 정자 생존율, 운동성(미토콘드리아 검사), DNA 손상(염색질인 크로마틴 구조) 검사를 위해 형광 염색을 한 후 성상검사를 FACS Aria 유세포 분석기를 이용해 실시하였다. 이때, Androhep (A)의 구성성분은 100 ㎖당 glucose 0.26 g, Sodium citrate 0.8 g, EDTA 0.24 g, Sodium bicarbonate 0.12 g, HEPES 0.9 g, Bovine Serum Albumin 25 ㎎을 함유하고 있다. 또한, Beltsville Thowing Solution (B)의 구성성분은 증류수 100 ㎖당 glucose 0.37 g, Sodium citrate 0.6 g, EDTA 0.125 g, Sodium bicarbonate 0.125 g, potassium chloride 75 ㎎을 함유하고 있다. 각 테스트를 이하에서 보다 상세히 설명한다.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.
<시험예 1> 정자 생존율 테스트Test Example 1 Sperm Survival Test
정자세포의 생존율 검사는 200 x 현미경상에서 기본적인 관찰을 실시한 후, 보다 객관적이고 정확한 분석을 위하여 생사염색을 실시하여 유세포 분석기 (flowcytometer)로 분석하였다. 구체적으로, 희석된 정자세포의 각 군을 보존시간 경과별로 SYBR-14 형광염료{살아있는 세포는 블루 레이저 광원(488 ㎚)에 의해 녹색의 형광을 발광하며 530/30 ㎚ 밴드필터에 의하여 형광 시그널이 집광됨}와 propidium iodide 형광염료{사멸된 세포는 블루 레이저 광원(488 ㎚) 에 의해 빨강색의 형광을 발광하며 610/20 ㎚ 밴드필터에 의하여 형광 시그널이 집광됨}을 5분간 실온에서 염색하여. 유세포 분석기에 옮겨 평균 20,000만개의 정자에서 녹색 및 빨강색 형광 비율을 분석하여 보존 시간 경과별 정자의 생존비율을 분석하였다(도 1).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 dyes (the dead cells emit red fluorescence by a blue laser light source (488 nm) and a fluorescent signal is collected by a 610/20 nm band filter) for 5 minutes at room temperature. . 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).
그 결과, 보존 후 처음 4일간 정자 생존율에는 3개 그룹에서 유의적인 차이가 인정되지 않았으나, 희석 보존 경과 후 5일부터는 A 및 B 제품군은 생존율이 저하되기 시작하였고, 특히 B 제품에서의 생존율 저하가 두드러지게 나타났다. 반면 본 발명의 희석제인 Q에서는 보존 경과 후 10일까지 비교적 안정되게 처음 생존율을 유지하는 것으로 나타났다.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 dilution preservation, the A and B family began to degrade, especially in the B product. Noticeably. On the other hand, Q, the diluent of the present invention, was shown to maintain the initial survival rate relatively stably until 10 days after the preservation.
<시험예 2> 정자 운동성 테스트Test Example 2 Sperm Motility Test
정자 활력 검사를 위해 정자의 에너지원인 정자 내 미토콘드리아 활성을 조사하였다. 구체적으로, 200 x 현미경하에서 기본적인 활력을 관찰 한 후 보다 정확하고 객관적인 활력 검사를 위해 미토콘드리아 막을 특정하게 염색할 수 있는 Rho-damine-123(미토콘드리아 기능이 정상인 세포는 블루 레이저 광원(488 ㎚) 에 의해 녹색형광을 발광하고 575/26 ㎚ 파장영역에서 형광 시그널이 집광됨) 형광 염료와 아울러 사멸한 정자를 선별하기 위하여 propidium iodide 형광염료를 각각 실온에서 10분간 염색하여 정자내 미토콘드리아 기능을 평균 20,000개의 정자를 유세포 분석기를 이용하여 보존 시간경과별로 비교 검사하였다(도 2).For sperm vitality test, we investigated mitochondrial activity in sperm, which is the energy source of sperm. Specifically, Rho-damine-123 (the cells with normal mitochondrial function are treated with a blue laser light source (488 nm), which can specifically stain mitochondrial membranes for more accurate and objective vitality testing after observing basic vitality under a 200 x microscope. In order to screen green fluorescence and to collect fluorescence signal in the wavelength range of 575/26 ㎚) In order to select dead sperm together with fluorescent dye, propidium iodide fluorescent dyes were stained for 10 minutes at room temperature to average 20,000 sperm in sperm Was compared by preservation time using a flow cytometer (Fig. 2).
그 결과, 정자 운동성은 정자 생존율 검사에서와 같은 유사한 패턴을 나타남을 확인하였다. 전 시험기간을 통해 Q는 정자 운동성에 큰 변화가 없이 유지하고 있어 10 일 이상 보존된 정자를 이용하여 인공수정을 실시하였을 때에도 수태율에 문제가 없음을 시사하고 있다.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.
<시험예 3> 정자 DNA 손상 테스트Test Example 3 Sperm DNA Damage Test
보존 경과 일수에 따른 정자세포막 지질의 산화로 발생할 수 있는 정자 DNA 손상을 검사하였다. 구체적으로, 정상적인 2중 나선구조의 DNA 가닥은 Acridine Orange 형광 염료에 의해 염색이 되고 블루 레이저 광원(488 ㎚) 에 의해 녹색의 형광을 발광하게 되어 530/30 ㎚ 밴드 필터에 의해 형광 시그널이 집광되는 반면 손상된 단일 가닥의 DNA 구조는 블루 레이저 광원(488 ㎚)에 의해 빨강 형광을 발광하게 되어 610/20 ㎚ 밴드 필터에 의해 형광 시그널이 집광된다. 상기 열거한 두 개의 형광염료를 실온에서 각각 5분간 염색한 후 평균 20,000개의 정자에서 보존 시간 경과별 형광 비율을 유세포 분석기로 비교, 분석하였다(도 3).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).
그 결과, 보존 후 7일 경과부터 A 및 B 제품군에서 2중 나선구조의 DNA 가닥에 대한 손상이 발생함을 보여주고 있고, 이는 현재 알려진 정자 크로마틴 구조의 변화와 남성 불임 간에 상관관계가 높다는 보고(D'Occhio et al., 2007. Biology of sperm chromatin structure and rela-tion-ship to male fertility and embryonic survival. Anim Reprod Sci. 101(1-2):1-17)에 비추어 보아 이들 제품군에 보존된 정자로 인공수정을 실시할 경우 수태율 저하를 가져올 수 있음을 시사한다. 그에 반하여, Q 군에서는 거의 안정되게 발광을 나타내어 상당한 기간 경과후에도 안정된 수태율을 유지시킬 수 있음을 나타낸다.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 rela-tion-ship to male fertility and embryonic survival.Anim Reprod Sci. 101 (1-2): 1-17) conserved in these families Implementing artificial insemination with sperm may impair the conception rate. In contrast, the Q group shows almost stable light emission, indicating that stable conception rate can be maintained even after a significant period of time.
<시험예 4> 정자 산화도 테스트Test Example 4 Sperm Oxidation Test
보존 경과 일수에 따른 정자 산화 정도를 측정하기 위하여 pH 변화를 pH 미터를 이용하여 희석하지 않은 원정액을 포함하여 제품별로 희석 보존된 정액 각 5 ㎖을 취하여 보존 시간 경과별로 비교 측정하였다(도 4).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). .
그 결과, 채취한 후 보존액에 희석하지 않은 원 정액은 예상대로 지질 대사로 인해 4일 이후부터는 급격한 산화 현상을 나타냈으며, A군은 4일 이후부터 산성쪽으로 하강하기 시작하였고, 반면에 B군은 오히려 pH가 알칼리성으로 올라가는 현상이 발생했으며(대사 증진으로 인한 일시적으로 활력의 증가를 가져오나 결국 짧은 시간 안에 활력의 급격한 저하를 가져온다), Q군은 비교적 전 기간을 통하여 pH가 유지되어 항산화 효과가 탁월함을 확인하였다.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.
지금까지 보관이 곤란한 돼지 액상정액을 장기보존할 수 있어서 혈통이 우수한 돼지의 품종을 보존할 수 있고, 돼지의 정액을 적절한 시기 및 장소에서 암돼지에 인공수정하여 수태율을 높일 수 있다.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 increase the fertility rate by artificial insemination of pig semen at the appropriate time and place.

Claims (9)

  1. 2개의 당(sugar)이 결합된 항산화 효과가 있는 플라보노이드류(flavonoids)의 일종인 쿼세틴(quercetin) 배당체 제재를 포함하는 돼지 액상정액 장기보존 희석제.A liquid long-term preservation diluent for pig liquids containing a quercetin glycoside preparation, which is a kind of antioxidant flavonoids in which two sugars are combined.
  2. 제 1항에 있어서, 상기 쿼세틴 배당체는 하기 구조식 1과 같이 3-glucoside-7-glucuronide가 결합된 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.According to claim 1, wherein the quercetin glycoside is a liquid liquid long-term preservation diluent of pig liquid, characterized in that 3-glucoside-7-glucuronide is bonded as shown in the following structural formula 1.
    (구조식 1)(Formula 1)
    Figure PCTKR2009003788-appb-I000002
    Figure PCTKR2009003788-appb-I000002
  3. 제 1항에 있어서, 상기 쿼세틴 배당체는 식물로부터 추출되는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.The long-term preservation diluent of pig liquid sperm of claim 1, wherein the quercetin glycoside is extracted from a plant.
  4. 제 1항에 있어서, 상기 쿼세틴 배당체는 증류수 100 ㎖ 당 0.01 내지 10 ㎎을 함유하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.According to claim 1, wherein the quercetin glycosides long term liquid preservation diluent of pig liquid, characterized in that it contains 0.01 to 10 mg per 100 ml of distilled water.
  5. 제 1항에 있어서, 상기 희석제는 쿼세틴 배당체 이외에 포도당, Sodium citrate, Sodium bicarbonate, EDTA, Potassium chloride, Potassium phosphate, Citric acid, Bovine Serum Albumin(Fraction V), glycine, Tris, 및 Trehalose로 구성된 기본용액을 포함하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.The diluent 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 quercetin glycosides. Pig liquid sperm long-term preservation diluent comprising a.
  6. 제 5항에 있어서, 상기 기본용액은 증류수 100 ㎖ 당 포도당 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 ㎎, glycine 0.5~3 g, Tris 0.5~30 g 및 Trehalose 0.01~1 g을 포함하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.The method of claim 5, 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. 제 5항에 있어서, 상기 기본용액은 추가적으로 항생제로 겐타마이신(gentamycin) 네오마이신(neomycin), 및 아프라마이신(apramycin)으로 구성된 군중에서 선택된 항생제를 함유하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.[6] The long-term preservation diluent for swine liquid semen of claim 5, wherein the base solution further contains an antibiotic selected from the group consisting of gentamycin neomycin and apramycin as an antibiotic. .
  8. 제 4항, 제 6항 및 제 7항 중 어느 한 항에 있어서, 상기 희석제는 증류수 100 ㎖ 당 쿼세틴 배당체 0.01 내지 10 ㎎, 포도당 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 ㎎, glycine 0.5~3 g, Tris 0.5~30 g, Trehalose 0.01~1 g 및 겐타마이신 50~10,000 IU를 포함하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.The method according to any one of claims 4, 6 and 7, wherein the diluent is 0.01 to 10 mg of quercetin glycoside per 100 ml of distilled water, 1 to 10 g of glucose, 0.2 to 3 g of sodium citrate, and 0.05 to 1 of sodium bicarbonate. 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 pig liquid semen long-term preservation diluent comprising a.
  9. 제 1항에 있어서, 상기 희석제는 10 내지 22℃에서 돼지 액상정액을 처리하는 것을 특징으로 하는 돼지 액상정액 장기보존 희석제.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 ℃.
PCT/KR2009/003788 2008-07-10 2009-07-10 Diluent for long-term preservation of liquid boar sperm WO2010005260A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020080066852A KR100903581B1 (en) 2008-07-10 2008-07-10 Porcine liquid semen diluent for long term preservation
KR10-2008-0066852 2008-07-10

Publications (2)

Publication Number Publication Date
WO2010005260A2 true WO2010005260A2 (en) 2010-01-14
WO2010005260A3 WO2010005260A3 (en) 2010-06-10

Family

ID=40982929

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2009/003788 WO2010005260A2 (en) 2008-07-10 2009-07-10 Diluent for long-term preservation of liquid boar sperm

Country Status (2)

Country Link
KR (1) KR100903581B1 (en)
WO (1) WO2010005260A2 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103081892A (en) * 2011-10-27 2013-05-08 北京清美联创干细胞科技有限公司 Frozen stock solution used for preserving adult stem cells for long time
CN104322483A (en) * 2014-10-09 2015-02-04 广东中农联生物制药有限公司 Boar antiviral long-acting semen diluent formula and preparation method
CN110301431A (en) * 2019-07-17 2019-10-08 西北民族大学 A kind of non-animal derived property sheep sperm cryo-conservation dilution and method
CN110367240A (en) * 2019-07-17 2019-10-25 西北民族大学 A kind of sheep sperm cryo-conservation dilution and method
US10470798B1 (en) 2018-11-30 2019-11-12 Ohana Biosciences, Inc. Methods for promoting fertilization
CN114097769A (en) * 2021-12-08 2022-03-01 山东道和生物科技有限公司 Pig semen dilution powder and processing technology thereof
CN115245161A (en) * 2022-08-17 2022-10-28 中国农业大学 Oregano oil-based long-acting diluent for normal-temperature preservation of boar semen
CN115251042A (en) * 2022-09-08 2022-11-01 安徽科技学院 Poultry semen dilution buffer solution, preparation method thereof and poultry semen cold storage method
CN116491498A (en) * 2023-04-27 2023-07-28 西北农林科技大学 Application of quercetin as diluent for goat semen low-temperature preservation

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101403597B1 (en) 2012-11-28 2014-06-03 대한민국 Composition for cryopreservation of semen
KR101546487B1 (en) 2013-06-28 2015-08-25 대한민국 Composition for cryopreservating sperm comprising LDL and anti-oxidant and uses thereof
KR101787007B1 (en) * 2016-12-08 2017-10-18 전북대학교산학협력단 A composition comprising flavonoid derivatives for treating and preventing Male Infertility
KR101787008B1 (en) * 2016-12-08 2017-10-20 전북대학교산학협력단 A composition comprising the combination of flavonoid derivatives and iridoid derivatives for treating and preventing Male Infertility
CN107637589A (en) * 2017-11-21 2018-01-30 广东中农联生物制药有限公司 Diluent of Pig's Spermatic Fluid and its production method
CN109258623A (en) * 2018-09-18 2019-01-25 华中农业大学 A kind of diluting boar semen agent and application
KR102316634B1 (en) 2019-03-25 2021-10-25 중앙대학교 산학협력단 Method for increasing female conception rate using a new seminal conservative solution

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR940004838B1 (en) * 1991-07-12 1994-06-02 주식회사 태평양 Method of extracting flavor from green tea
US20020164795A1 (en) * 1999-06-02 2002-11-07 Shokyu Gen Storage agent for preservation of an animal cell, tissue or organ, and preserved process of the same
KR20070018145A (en) * 2004-07-23 2007-02-13 더 프록터 앤드 갬블 캄파니 Skin care composition containing a flavonoid and vitamin ??

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR940004838B1 (en) * 1991-07-12 1994-06-02 주식회사 태평양 Method of extracting flavor from green tea
US20020164795A1 (en) * 1999-06-02 2002-11-07 Shokyu Gen Storage agent for preservation of an animal cell, tissue or organ, and preserved process of the same
KR20070018145A (en) * 2004-07-23 2007-02-13 더 프록터 앤드 갬블 캄파니 Skin care composition containing a flavonoid and vitamin ??

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
APPLETON J. NATURAL MED. J. vol. 2, no. 1, January 2010, pages 1 - 6 *
PARK H. -J. ET AL. J. KOR. SOC. HORT. SCI. vol. 45, no. 3, June 2004, pages 138 - 142 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103081892B (en) * 2011-10-27 2014-04-09 北京清美联创干细胞科技有限公司 Frozen stock solution used for preserving adult stem cells for long time and preparation method thereof
CN103081892A (en) * 2011-10-27 2013-05-08 北京清美联创干细胞科技有限公司 Frozen stock solution used for preserving adult stem cells for long time
CN104322483A (en) * 2014-10-09 2015-02-04 广东中农联生物制药有限公司 Boar antiviral long-acting semen diluent formula and preparation method
US10470798B1 (en) 2018-11-30 2019-11-12 Ohana Biosciences, Inc. Methods for promoting fertilization
US10603075B1 (en) 2018-11-30 2020-03-31 Ohana Biosciences, Inc. Compositions and methods for enhancing sperm function
CN110301431A (en) * 2019-07-17 2019-10-08 西北民族大学 A kind of non-animal derived property sheep sperm cryo-conservation dilution and method
CN110367240A (en) * 2019-07-17 2019-10-25 西北民族大学 A kind of sheep sperm cryo-conservation dilution and method
CN114097769A (en) * 2021-12-08 2022-03-01 山东道和生物科技有限公司 Pig semen dilution powder and processing technology thereof
CN115245161A (en) * 2022-08-17 2022-10-28 中国农业大学 Oregano oil-based long-acting diluent for normal-temperature preservation of boar semen
CN115251042A (en) * 2022-09-08 2022-11-01 安徽科技学院 Poultry semen dilution buffer solution, preparation method thereof and poultry semen cold storage method
CN115251042B (en) * 2022-09-08 2023-06-02 安徽科技学院 Poultry semen dilution buffer solution and preparation method thereof and poultry semen cooling method
CN116491498A (en) * 2023-04-27 2023-07-28 西北农林科技大学 Application of quercetin as diluent for goat semen low-temperature preservation
CN116491498B (en) * 2023-04-27 2024-04-23 西北农林科技大学 Application of quercetin as diluent for goat semen low-temperature preservation

Also Published As

Publication number Publication date
WO2010005260A3 (en) 2010-06-10
KR100903581B1 (en) 2009-06-22

Similar Documents

Publication Publication Date Title
WO2010005260A2 (en) Diluent for long-term preservation of liquid boar sperm
Thuwanut et al. The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa
Andrabi et al. Effect of non-enzymatic antioxidants in extender on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa.
Isaak et al. Manitoba lingonberry (Vaccinium vitis-idaea) bioactivities in ischemia-reperfusion injury
WO2010137878A9 (en) Plant stem cell derived from cambium of family gingkoaceae and method for the isolation thereof
CN101023966A (en) Green algal extract containing astaxanthin having high storage stability
US20120264105A1 (en) Composition for Preserving Reproductive Cells and Method of Using
CN111771873B (en) Method for improving quality of thawed pig sperms
Ahmed et al. Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa
CN113317312B (en) Diluent preparation capable of prolonging normal-temperature survival period of boar semen
Lahnsteiner et al. Investigations on the metabolism of viable and nonviable gilthead sea bream (Sparus aurata) eggs
Hu et al. Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing
Hegazi In vitro studies on Delonix elata L., an endangered medicinal plant
WO2021132655A1 (en) Inhibitor of fertilized egg fragmentation
KR20070079465A (en) Fermented extract of citrus sunkii hort, method for processing thereof, and use
Ren et al. Effects of Isatis root polysaccharide on boar sperm quality during liquid storage and in vitro fertilization
Mohsin et al. Effect of adding antioxidants to Tris extender on cooling and post-cryopreservative semen characteristics of Holstein bulls 2. Salvia officinalis L aqueous extract.
Mohammadi et al. Effects of hydroethanolic extracts of Terminalia chebula and Thymbra spicata on ram fresh semen under normal and oxidative stress conditions
Vizzarri et al. Lippia citriodora (verbascoside) extract supplementation: Effect on rabbit semen quality in vivo and in vitro
CN115119833B (en) Semen cryopreservation diluent and application thereof
NL2034272A (en) Long-acting diluent for boar semen preservation at normal temperature based on oregano essential oil
Martins et al. Antioxidant effect on viability of boar semen cooled to 15° C
Eid Dietary grape pomace affects lipid peroxidation and antioxidative status in rabbit semen.
Tsai et al. Studies on cryoprotectant toxicity to early stage zebrafish (Danio rerio) ovarian follicles
CN115624024B (en) Application of codonopsis pilosula polysaccharide as additive in sheep semen low-temperature diluent

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09794666

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09794666

Country of ref document: EP

Kind code of ref document: A2