KR102054710B1 - Medium composition for in vitro fertilization comprising granule from Acer mono sap as effective component and method of in vitro fertilization using the same - Google Patents

Abstract

The present invention relates to an in vitro fertilization medium composition for mammalian eggs and sperm containing grouse sap granules as an active ingredient, and an in vitro fertilization method using the same. It can be used for breeding techniques.

Description

Medium composition for in vitro fertilization comprising granule from Acer mono sap as effective component and method of in vitro fertilization using the same}

The present invention relates to an in vitro fertilization medium composition comprising grouse sap granules as an active ingredient and an in vitro fertilization method using the same.

Acer mono (Acer pictum subsp. Mono) is much distributed in the altitude of 100 ~ 1,800 m of the leaves such as the large broad-leaved tall tree of Araliaceae Jiri, White Clouds Mountain, mouth acid, or Jogyesan Kangwon Province shed leaves. The origin of Gororo is derived from the Chinese word Goliasu, which is good for bones. The sap collected from Goryeo tree is known to be effective in diuresis, constipation, gastrointestinal diseases, neuralgia, gout, and postpartum pain. In addition, Acer mono sap has a high calcium content and contains many minerals such as magnesium and potassium that affect muscles and nerves in ionic form, resulting in high body absorption rate, blood sugar control, fatigue recovery, health, and physical strength enhancement. It is known to be effective. Gororap sap contains the most useful nutritional ingredients and functionalities, so it is most used than sap of other species (such as maple, birch, etc.). This is low.

In mammals, normal fertilization refers to the infiltration of a single spermatozoa into a matured ovum, forming a single female pronucleus and a male pronucleus, each of which is called the paternal and maternal This means that the chromosomes are normally arranged to develop into embryos.

In vitro fertilization of mammals is a very useful technique for breeding and improving livestock. Various assisted reproductive technologies, such as in vitro fertilization, have been implemented to promote the animal industry and treat human infertility or infertility, but their effectiveness is not yet high. In order to establish in vitro fertilization conditions that can increase the in vitro fertilization rate of mammals, various studies related to oxygen concentration, presence or absence of oocytes, and embryonic development rate according to egg size difference have been conducted. The unknown developmental factors that optimize the in vitro fertilization rate are not fully understood. Therefore, there is a need for a method that can be applied to the assisted breeding technology of mammals by improving the IVF rate by establishing more improved IVF conditions.

Meanwhile, Korean Laid-Open Patent Publication No. 2012-0033932 describes a method for storing blast furnace sap using ultrafiltration, and Korean Patent No. 1851575 discloses an in vitro fertilization comprising 'diprotose dehydride 4' as an active ingredient. Media composition and in vitro fertilization method using the same ', but the in vitro fertilization medium composition comprising the grouse sap granules of the present invention as an active ingredient and the in vitro fertilization method using the same have not been described.

The present invention is derived from the above demand, the present inventors have confirmed that the sperm cultured in the medium to which the grouse sap granules are added, the motility is increased than the sperm cultured in the medium without the grouse sap granules, aging When the grouse sap granules were treated to sperm or sperm with reduced motility, it was confirmed that sperm vitality was increased. In addition, the present invention was completed by confirming that the total fertilization rate was increased when the oocytes and sperm were cultured in an in vitro fertilization medium containing grouse sap granules.

In order to solve the above problems, the present invention provides a medium composition for in vitro fertilization of mammalian eggs, sperm containing grouse sap granules as an active ingredient.

The present invention also provides a method for in vitro fertilization of mammalian eggs and sperm comprising the step of modifying the sperm and egg of the mammal in the in vitro fertilization medium composition.

In addition, the present invention comprises the steps of (a) collecting the follicle eggs from mammals other than humans; (b) recovering immature eggs from the follicles collected in step (a); (c) maturing the immature egg recovered in step (b) in vitro; (d) adding in vitro fertilized eggs and sperm of step (c) to the in vitro fertilization medium composition of the present invention for in vitro fertilization; And (e) culturing the in vitro fertilized fertilized in vitro fertilized in culture medium provides a method for mass production of mammalian in vitro fertilized egg.

The present invention also provides a composition for enhancing sperm motility in a mammal comprising groin sap granules as an active ingredient.

The present invention also provides a method for improving sperm motility in a mammal comprising treating the sperm motility composition to a sperm separated from the mammal.

In vitro fertilization medium composition comprising the grouse sap granules of the present invention can increase fertilization rate by improving the sperm motility and vitality of aged sperm, it is expected that it can be utilized in assisted breeding techniques of mammals.

1 is a result of measuring the motility of the pig sperm according to the treatment of groin sap granules. A; Motile sperm, B; Progressive sperm.
Figure 2 is the result of measuring the motility of the aged sperm of the pigs according to the treatment of grouse sap granules. A; Motile sperm, B; Forward motile sperm.
Figure 3 is the result of measuring the number of binding of sperm to the porcine egg cells in accordance with the treatment of grouse sap granules.
Figure 4 is the result of measuring the total fertilization rate of the in vitro fertilized egg in accordance with the treatment of grouse sap granules.

In order to achieve the object of the present invention, the present invention provides a medium composition for in vitro fertilization of mammalian eggs, sperm containing grouse sap granules as an active ingredient.

In the in vitro fertilization medium composition of the present invention, the croquette sap granules are prepared by mixing at least one powder selected from isomalt, maltodextrin, crystal sugar, or stevia. The core of the granules may be, but is not limited to, granules of the grouse sap granules formed by spraying the sap collected from the goro tree sap, while the particles of the goro sap surround the nucleus of the granules.

In addition, the groyne sap granules of the present invention can significantly reduce the volume than the sap or syrup form product by transforming the groyne sap into a granular form, it is possible to increase the shelf life, easy to store and transport, powder that is well soluble in water It has a merit that it has high solubility (Korean Patent No. 1603937).

The term "in vitro fertilization" of the present invention is a method distinguished from the state of fertilization of the egg and sperm in the body, and means a series of laboratory processes to fertilize and culture in conditions similar to the environment of the uterus in the laboratory incubator (incubator) The medium composition of the present invention is a medium composition optimized for such in vitro fertilization.

The in vitro fertilization medium composition of the present invention can be used by adding the groin sap granules to the basal medium used for conventional mammalian in vitro fertilization.

In the present invention, the term "basic medium" is different depending on the species of mammal, but the medium for the early mammalian embryo culture medium containing inorganic salts, carbon sources, amino acids, bovine serum albumin and cofactors, etc. Means, and may include all common common media well known to those skilled in the art.

The inorganic salt may be at least one selected from sodium chloride (NaCl), potassium chloride (KCl), and sodium bicarbonate (NaHCO ₃ ), but is not limited thereto.

The carbon source may be one or more selected from glucose, sodium pyruvate, and calcium lactate, but is not limited thereto.

The amino acids are essential amino acids valine (leucine), leucine, isoleucine (isoleucine), methionine (methionine), threonine (threonine), lysine (lysine), phenylalanine (phenylalanine), tryptophan, histidine ); Quasi-essential amino acids glutamine, glycine, cysteine, arginine, tyrosine, proline; And non-essential amino acids alanine, aspartic acid, glutamic acid, and asparagine, but are not limited thereto.

The cofactors are zinc (Zn), copper (Cu), manganese (Mn), selenium (Se), cobalt (Co), molybdenum (Mo), vanadium (V), iodine (I), bromine (Br), fluorine ( F), at least one selected from nickel (Ni), silicon (Si), tin (Su), chromium (Cr), boron (B), buffers and antibiotics, but is not limited thereto.

The basal medium is a commercially available medium known for mammalian cell culture, for example, mTBM (modified Tris-buffered medium; Abeydeera LR. Et al., Theriogenology, 48 (4): 537-544, 1997) , PZM-3 (Pocrine zygote medium-3; Yoshioka et al., Biology of reproduction, 66 (1): 112-119, 2002), NCSU-23 (North Carolina State University medium; Petters and Wells, J. Reprod. Fert., Suppl. 48: 61-73, 1993), CR1-aa (Rosenkrans, CF Jr. et al., Biol. Reprod., 49 (3): 459-462, 1993), TCM (tissue culture medium of medium 199; Yue, C. et al., Development, 121: 2645-2654, 1995), CZB (Chatot-Ziomek-Bavister medium; Chatot, CL et al., J. Reprod. Fert., 86: 679-688 , 1989) and the like, but is not limited thereto. The commercialized medium may be used as it is or may be partially modified.

The in vitro fertilization medium composition according to the present invention may include the grouse sap granules at a concentration of 50 to 250 μg / ml, preferably at a concentration of 100 to 200 μg / ml, but is not limited thereto.

In vitro fertilization medium composition of the present invention includes the grouse sap granules to enhance the sperm motility as an active ingredient has the effect of increasing the total fertilization rate in vitro fertilization conditions.

In the in vitro fertilization medium composition of the present invention, the mammal may include animals other than humans, for example, live animals such as pigs, cows, sheep, horses, pets, and experimental animals such as mice, guinea pigs, and the like. Preferably it may be a pig, but is not limited thereto.

The present invention also provides a method for in vitro fertilization of mammalian eggs and sperm comprising the step of modifying the sperm and egg of the mammal in the in vitro fertilization medium composition.

The in vitro fertilization method of the present invention can increase the in vitro fertilization rate of mammalian eggs and sperm by using an in vitro fertilization medium composition comprising grouse sap granules which increase motility of sperm as an active ingredient.

Oocytes and sperm of mammals to which the in vitro fertilization method of the present invention can be applied are experimented with eggs and sperm of animals other than humans, for example, livestock such as pigs, cows, sheep, horses, pets, and rats and guinea pigs. It may be an egg and a sperm of an animal, preferably an egg and a sperm of a pig, but are not limited thereto. Moreover, as long as an egg and a sperm which can be fertilized in vitro are not specifically limited to the kind.

The present invention also provides

(a) harvesting follicular eggs from mammals other than humans;

(b) recovering immature eggs from the follicles collected in step (a);

(c) maturing the immature egg recovered in step (b) in vitro;

(d) adding in vitro fertilized eggs and sperm of step (c) to the in vitro fertilization medium composition of the present invention for in vitro fertilization; And

(e) culturing the fertilized in vitro fertilized embryo in vitro culture medium provides a method for mass production of mammalian in vitro fertilized egg.

The present invention also provides a composition for enhancing sperm motility in a mammal comprising groin sap granules as an active ingredient. The composition according to the present invention contains groin sap granules to increase the motility of the sperm as an active ingredient, the sperm motility can be increased by adding the composition to the medium containing sperm cells.

The sperm motility (%) of sperm was cultured after sperm was cultured in the grouse sap added medium of the present invention, and it was confirmed that the sperm motility was significantly higher than that of the grouse sap granules.

The composition for improving sperm motility according to the present invention may include groust sap granules at a concentration of 50 to 250 μg / ml, and preferably at a concentration of 100 to 200 μg / ml, but is not limited thereto.

The present invention also provides a method for enhancing sperm motility in a mammal comprising treating the sperm motility composition to a sperm separated from the mammal.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Preparation Example 1 Preparation of In Vitro Fertilization Medium for Pigs Treated with Grooved Sap Granules

Medium for in vitro fertilization was modified tris-buffered medium (mTBM) with 0.2% bovine serum albumin (BSA); NaCl 113.1 mM, KCl 3 mM, CaCl ₂ · 2H ₂ O 7.5 mM , Tris 20.0 mM, glucose 11.0 mM, sodium pyruvic acid 5.0 mM, caffeine 3.4 mM) was prepared by the addition of gorro sap granules (Maple granule, Kasodom, Korea) to 100 or 200 ㎍ / ㎖ concentration.

Preparation Example 2 Preparation of In Vitro Matured Follicle Egg and Sperm of Pig for In Vitro Fertilization

The ovaries of slaughtered piglets were slaughtered at the slaughterhouse, and the extracted ovaries were transferred to a laboratory in 0.9% saline. Immature eggs attached to follicular fluids and cumulus cells were collected by aspiration from 2-6 mm diameter follicles using a 10 ml syringe equipped with an 18-gauge needle. In the collected follicles, only the immature oocytes with uniformly adherent and healthy cytoplasm were selected and washed with TL-HEPES (Tyrode lactate-Hepes) medium containing 0.01% polyvinyl alcohol (PVA). In vitro maturation for 44 hours in tissue culture medium (TCM) -199 supplemented with follicle stimulating hormone (FSH), luteinizing hormone (LH) and epidermal growth factor (EGF) In vitro maturation (IVM) was induced. 0.1% hyaluronidase was applied to the matured oocytes to remove the oocytes surrounding the oocytes, and then the oocytes of the medium phase (metaphase II) where the cytoplasm is healthy and the first polar body protrudes. Only bays were selected.

In addition, the liquid pig liquid of Example 1 was washed three times with a PBS solution containing 0.01% polyvinyl alcohol to obtain a pellet of the liquid liquid, the pellet was suspended in mTBM medium and used in the experiment.

Example 1 Analysis of Motility of Swine Sperm by Treatment of Grooved Sap Granules

To determine the effect of groin sap granules on the sperm motility of pigs, the liquid sperm preservation solution BTS (beltsville) after checking the motility (> 85% vigor) and concentration (sperm concentration) of pig sperm collected from pig farms thawing solution; 1x10 ⁸ spermatozoa water / ml in 3.7 g glucose, 100 g distilled water, 0.6 g sodium citrate, 0.125 g sodium bicarbonate, 0.125 g EDTA, 0.075 g potassium chloride, 0.06 g penicillin G, 0.1 g streptomycin) The semen was diluted to a concentration of liquid to prepare a liquid semen (liquid semen) and stored in 17 ℃ storage for 3 days was used for the experiment. Then, gorogo sap granules were treated in liquid sperm by concentration (100 and 200 ㎍ / ml), and incubated for 3 hours at 37.5 ° C, followed by sperm motility with a computer-assisted sperm analysis (Microptic, Spain) program. As a control, sperm in the liquid sperm was not treated with grouse sap granules.

As a result, it was confirmed that the motility sperm (% motile sperm) was increased in the Goroap sap granules treatment group (90.4%) compared to the control (82.9%), especially (100.g / ml) 1A). In addition, the motility sperm (% progressive sperm) was also increased in the grouse sap granules treatment compared to the control, it was confirmed that there is no significant difference (Fig. 1B).

Example 2 Analysis of Motility of Aged Sperm in Pigs Treated with Grooved Sap Granules

After storing the pig liquid sperm prepared in Example 1 for 10 days in a 17 ℃ storage cell treated with gorgo sap granules by concentration (100 and 200 ㎍ / ㎖) to the liquid sperm, incubated for 3 hours at 37.5 ℃ The motility of the aged sperm was then observed using a computer-assisted sperm analysis (Captic, Spain) program, and the control group was aged sperm in liquid sperm without grouse sap granules.

As a result, the motility sperm (% motile sperm) was significantly increased in the treatment group (90.7% and 91.7%, respectively) of the grouse sap granules compared to the control (79.6%) (Fig. 2A), forward sperm (% progressive sperm) ) Was also increased in the groin sap granules treatment group compared to the control, it was confirmed that there is no significant difference (Fig. 2B). Through this, it could be inferred that the groin sap granules of the present invention can increase the vitality of aged sperm or motility deteriorated sperm.

Example 3 Determination of the Number of Sperm Bound to Porcine Oocytes

10 oocytes selected in Preparation Example 2 and sperm diluted in mTBM medium were injected into the IVF medium treated with concentrations (100 and 200 μg / ml) of grouse sap granules at a concentration of 1 × 10 ⁵ sperm / ml. Co-incubated for 30 minutes in an incubator at 38.5 ° C. and 5% CO ₂ , harvested, fixed with 2% formaldehyde and fixed with 2.5 mg / mL DAPI (4 ′, 6-diamidino). -2-phenylindole) was used to observe the number of sperm attached to the zona pellucida (ZP) of the egg using a fluorescence microscope.

As a result, the number of sperm attached to the zona pellucida of the egg was significantly increased in the treatment groups (7.7 and 7.9, respectively) by concentration of the grouse sap granules than the untreated control (4) of the grouse sap granules (Fig. 3). . Through this, it can be inferred that sperm invasion rate can be increased when sperm and eggs are cultured in the in vitro fertilization medium to which the grouse sap granules of the present invention are added.

Example 4 Fertilization Rate Analysis of Pig Sperm and Egg

In vitro fertilization in which the egg selected in Preparation Example 2 and 1 μl of sperm diluted in mTBM medium (final concentration 0.5 × 10 ⁵ sperm water / ml) were treated by concentrations of granule sap granules (100 and 200 μg / ml) Injected into the medium, respectively, and co-incubated for 3 hours in an incubator at 38.5 ° C. and 5% CO ₂ conditions to induce IVF, and then the in vitro fertilized egg was incubated with PZM-3 (porcine). zygote medium-3) and incubated for 16-20 hours. After incubation, the embryos were fixed with 2% formaldehyde and stained with 2.5 mg / ml DAPI (4 ', 6-diamidino-2-phenylindole). After observing with a fluorescence microscope to analyze the fertilization rate.

As a result, the total fertilization rate was significantly increased in the treatment group (76% and 69.9%, respectively) of the Goroque sap granules compared to the control (51.1%), especially in the Goroque sap granules treatment of 100 ㎍ / ㎖ concentration It was confirmed (FIG. 4).

Through the above results, it can be inferred that treatment of grouse sap granules in the medium during in vitro fertilization enhances sperm motility and facilitates invasion of sperm into eggs, thereby increasing fertilization rate. Therefore, the grouse sap granules of the present invention can be used to prepare in vitro fertilization, artificial insemination, dilution of copper crystals and the like in mammals.

Claims (9)

An in vitro fertilization medium composition for mammalian oocytes and sperm comprising grouse sap granules as an active ingredient. The medium composition for in vitro fertilization of mammalian eggs and sperm according to claim 1, wherein the medium composition further comprises at least one selected from inorganic salts, carbon sources, amino acids and bovine serum albumin. The medium composition for in vitro fertilization of mammalian eggs and sperm according to claim 1, wherein the medium composition comprises groin sap granules having a concentration of 50 to 250 µg / ml. The in vitro fertilization medium composition for mammalian eggs and sperm according to claim 1, wherein the mammal is an animal other than a human. A method for in vitro fertilization of mammalian eggs and sperm, comprising the step of modifying by adding a sperm and an egg of a mammal to the in vitro fertilization medium composition according to any one of claims 1 to 4. (a) harvesting follicular eggs from mammals other than humans;
(b) recovering immature eggs from the follicles collected in step (a);
(c) maturing the immature egg recovered in step (b) in vitro;
(d) adding in vitro fertilized eggs and sperm of step (c) to the in vitro fertilization medium composition of any one of claims 1 to 4 in vitro fertilization; And
(e) culturing the in vitro fertilized egg fertilized in step (d) in a culture medium. A composition for enhancing sperm motility in a mammal comprising grouse sap granules as an active ingredient. According to claim 7, wherein the composition is a composition for improving sperm motility in mammals, characterized in that it comprises grouse sap granules of 50 ~ 250 ㎍ / ㎖ concentration. A method of enhancing sperm motility in a mammal comprising treating the composition of claim 7 with sperm isolated from the mammal.

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