KR101821575B1 - Medium composition for in vitro fertilization comprising difructose dianhydride 4 as effective component and method of in vitro fertilization using the same - Google Patents

Abstract

The present invention relates to a medium composition for external fertilization containing difructose dianhydride 4 as an active ingredient, and an external fertilization method using the same. The external fertilization method using the medium composition for external fertilization containing difructose dianhydride 4 as the active ingredient shows an increase in formation of normal fertilized egg compared to a case when medium compositions for external fertilization without difructose dianhydride 4 are used, while increasing development rate of blastocyst and division rate of produced fertilized egg.

Description

[0001] The present invention relates to a medium composition for in vitro fertilization comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, as an active ingredient, and a method for in vitro fertilization using the same.

The present invention relates to a culture medium composition for in vitro fertilization comprising Diffractosidanhydride 4 as an active ingredient and a method for in vitro fertilization using the same.

Traditionally, the method for improving seeds used in mammals other than humans is to breed livestock with excellent traits depending on crossing between individuals. This requires growing livestock until the time of crossing, so that it takes a long time to breed, And the speed was also very low. In order to solve the above problems, embryo transfer techniques derived from individuals having excellent traits have been developed.

To date, ovulation treatment techniques have been used to mass-produce fertilized eggs for embryo transfer system. The production of embryos by superovulation treatment was induced by follicular stimulation hormone (FSH) or gonadal serum gonadotropin (PMSG) at the time of induction of a number of follicles and ovulation, Surgical or non-surgical method. However, there is a problem that the ovarian hyperstimulation method should be improved in economy and simplicity in which a certain abstinence period should be set after the use of the expensive hormone agent and use thereof.

 In order to solve the problems of the above-mentioned superovulation method, researches on the production technology of embryo by in vitro fertilization and cultivation have been carried out. Co-culture system with somatic cells such as tubal epithelial cells, uterine fibroblasts and trophoblast cells has been widely used as the most general culture system for increasing the in vitro production efficiency of embryos. However, when somatic cells are co-cultured with embryos, somatic cells It has been pointed out that the metabolism of substances in the culture medium causes irregular effects such as promoting the development of the embryo or increasing the chance of contamination and suppressing the development of the embryo. In order to minimize the variation and maintain a certain level of development Simple cell culture system that excludes somatic cells was introduced to control the concentration of glucose, phosphate and amino acid in simple culture medium, polyvinyl alcohol (PVA), sodium bicarbonate, Simple cultivation for increasing fertilization rate of embryos by controlling the concentration of oxygen and controlling the oxygen partial pressure Various studies on the system have been attempted.

In general, normal fertilized eggs modified in vitro should undergo fertilization sufficiently before fertilization to reach the blastocyst stage for fertilization to the uterine bottom of the transgenic mammal.

This is because it contains enough protein enzymes necessary for implantation in the cells of the uterus in the exposed cells of the trophoblast, the cell membrane of the blastocyst.

Most in vitro fertilized embryos stop their ovulation at 4 to 8 cell cycles, making it impossible to implant at the bottom of the uterus of transgenic animals.

Therefore, it is important to secure intracellular trophoblast by increasing developmental rate of blastocyst in embryo.

In spite of the above-mentioned many studies, the unknown developmental factors that increase the development rate of the embryo developmental stage of the embryo are not completely clarified, and the conditions for the development of the improved embryo are established, There is a need for a method.

On the other hand, difructose dianhydride (DFA) is a compound first discovered by Jackson et al. (1929), produced during the production of fructose syrup by treatment of inulin with sulfuric acid, and two fructose The reducing end is a cyclic 2-sugar bonded to a non-reducing hydroxyl group at the end of fructose, which is different from each other. To date, five types of Diffractosidanhydride, DFA 1 to 5, have been known. Diprofloxacin can be used as a low-calorie sweetener in non-digestible, non-fructose-free disaccharides that do not degrade in the body of animals. It can be used as a bifidus microbial growth factor and inhibits the occurrence of cavities. In vivo minerals Absorption promoting factors and the like.

Polyfructan (fructose homopolymer), which is a natural raw material that can be used in the production of dipstick tosudan hydride 4, is inulin and levan, and can be chemically or enzymatically synthesized from them.

Among them, 2,6 ': 6,2' difructose dianhydride (DFA 4) is used as an active ingredient of levan fructosetransferase produced by microorganisms of the order Arthrobacter or Pseudomonas , LFTase), and Korean Patent No. 0380970 discloses a method for producing levofloxacin hydrate 4 by using levan fructo-transferase isolated from Astrobacterium urea fructus, .

Korean Patent No. 1107329 discloses a medium composition for improving embryo development rate and a method for mass production of a mammalian embryo using the same, and Korean Patent Publication No. 2011-0039689 Discloses a culture medium composition for in vitro culture of embryos and an in vitro culture method using the same, but the in vitro fertilization medium composition containing the present invention as an effective ingredient and the in vitro fertilization method using the same, There is no way.

The present invention has been made in view of the above-mentioned needs, and an object of the present invention is to provide a medium composition for in vitro fertilization comprising Diffractosidanhydride 4 as an active ingredient and a method for in vitro fertilization using the same, Sperm motility was increased in comparison with an in vitro fertilization medium composition in which pig sperm and egg were modified using an in vitro fertilization medium composition containing dianhydride 4, The present invention has been completed by confirming that it has an effect of reducing the generation of active oxygen species of the spermatogonium, increasing the sperm egg binding, increasing the generation of normal embryos, increasing the division rate of the generated embryos and the development rate of the blastocyst. Respectively.

In order to achieve the above object, the present invention provides a medium composition for in vitro fertilization of mammalian spermatozoa comprising dipstick tosudan hydride 4 as an active ingredient.

The present invention also provides a method for in vitro fertilization of a mammalian egg or sperm comprising the step of adding a sperm and an egg of a mammal to the culture medium for in vitro fertilization and then modifying the medium.

The present invention also provides a method for the treatment of ovarian cancer, comprising the steps of: (a) obtaining follicles from a mammal other than a human;

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

(c) in vitro maturation of the immature oocytes recovered in step (b);

(d) in vitro fertilization of oocytes and spermatozoa matured in vitro in step (c); And

(e) culturing the in vitro fertilized egg modified in the step (d) in a culture medium.

The present invention also provides a diluted liquid semen diluted with a liquid comprising 0.05 to 0.5% (w / v) of Difructose dianhydride 4 as an active ingredient.

The in vitro fertilization medium composition and the in vitro fertilization method using the same as the active ingredient of the present invention are superior to the conventional in vitro fertilization medium composition containing no Diffractosidan hydride 4, And the development rate of the blastocysts can be increased. Therefore, it can be usefully used for constructing a mass production system of improved quality in vitro fertilized embryos.

Therefore, the mass production method of mammalian fertilized eggs of the present invention can improve the embryo development and blastocyst quality of the in vitro fertilized embryo, which have remained insufficient yet, and can be effectively utilized for the efficient production of mammalian embryo transfer .

FIG. 1 is a graph showing the degree of sperm motility preservation according to the concentration of dipstick tosudan hydride 4. FIG. The mean is the standard error of mean (SEM) of 5 replicates, meaning that there is a statistically significant difference between a and b (p <0.05).
FIG. 2 is a graph showing the degree of production of reactive oxygen species (ROS) by sperm concentration according to the concentration of dipstick tosudan hydride. The mean value is the mean ± SEM of six replicates, indicating that there is a statistically significant difference between a, b, and c (p <0.05).
FIG. 3 is a graph showing the degree of oocyte binding of spermatozoa according to the concentration of Diffractosidone hydride 4. FIG. The number in parentheses at the top of the graph means the number of eggs. The mean value is the mean ± SEM of three replicates, indicating that there is a statistically significant difference between a and b (p <0.05).
FIG. 4 is a graph showing the fertilization rate of the IVF embryo according to the concentration of Diffractosidone hydride 4. FIG. The mean value is the mean ± SEM of three replicates, indicating that there is a statistically significant difference between a and b (p <0.05).
FIG. 5 is a graph showing the rate of blastocyst development and blastocyst release rate according to the concentration of Diffractosidone hydride 4. FIG. The mean value is the mean ± SEM of 5 replicates, meaning that there is a statistically significant difference between a, b, and c (p <0.05).
FIG. 6 is a graph showing the average number of cells per blastocyst according to the concentration of Diffractosidone hydride 4. FIG. The mean is the standard error of mean (SEM) of 5 replicates, meaning that there is a statistically significant difference between a and b (p <0.05).

The present invention relates to a medium composition for in vitro fertilization of a mammalian egg and sperm containing Diffractosidanhydride 4 as an active ingredient.

The term "in vitro fertilization" in the present invention refers to a series of laboratory processes in which an egg and sperm are separated from the state of fertilization in the body to modify and cultivate the same in a laboratory incubator under conditions similar to the environment of the uterus , The medium composition of the present invention is a medium composition optimized for such in vitro fertilization.

Sperm and eggs of mammals to which the inventive media composition can be applied are sperm and eggs of mammals other than humans, preferably livestock, more preferably pig sperm and egg. There are no particular restrictions on sperm and eggs that can be modified in vitro.

 The medium composition preferably contains 0.05-0.75% (w / v), more preferably 0.05-0.6% (w / v), even more preferably 0.05-0.15% of dipstick toxin hydride 4 (w / v), and most preferably 0.1% (w / v), but is not limited thereto.

The medium composition of the present invention can be used by adding diproxosidone hydride 4 to the base medium used for conventional mammalian in vitro fertilization. The term "basic medium" in the present invention is a culture medium for mammalian early embryonic cell culture which basically includes inorganic salts, carbon source, amino acid, bovine serum albumin (BSA) Lt; RTI ID = 0.0 &gt; well-known &lt; / RTI &gt;

The above-mentioned medium preferably includes at least one selected from inorganic salts such as NaCl, KCl and NaHCO ₃ , but is not limited thereto.

The carbon source of the culture medium is preferably, but not limited to, at least one selected from glucose, sodium pyruvate and calcium lactate.

The amino acid of the medium may be selected from the group consisting of essential amino acids valine, leucine, isoleucine, methionine, threonine, lysine, phenylalanine, tryptophan, Histidine and the nonessential amino acids cysteine, tyrosine, histidine, alanine, aspartic acid, glutamic acid, glycine, proline, ), Serine, but is not limited thereto.

The medium may be supplemented with at least one of zinc, copper, manganese, selenium, cobalt, molybdenum, vanadium, iodine, bromine, But is not limited to, at least one selected from fluorine (F), nickel (Ni), silicon (Si), tin (Su), chromium (Cr3 +), boron (B) .

As a preferred embodiment, the basic medium may include a plurality of commercialized mediums known for culturing mammalian cells, for example, NCSU-23 (North Carolina State University medium; Petters and Wells, J. Reprod. (TCM) (tissue culture medium of medium 199, Gibro (R)), CR1-aa (Rosenkrans, CF Jr., et al., Biol. Reprod., 49 (3), 459-462, , USA; Yue, C., et al., Development, 121: 2645-2654, 1995), CZB (Chatot-Ziomek-Bavister medium; Chatot, CL, et al., J. Reprod. -688, 1989), mTBM and the like can be used as they are. In the case of pigs, mTBM (modified Tris-buffered medium, Abeydeera LR., Et al., Theriogenology 48 (4): 537-544, 1997) 3 (Pocrine zygote medium-3, Yoshioka et al., Biology of reproduction 66 (1): 112-119, 2002).

In the present invention, the term "blastocyst" refers to a blastocyst formed by a blastocyst after densely packed embryo, and the inner cell mass to differentiate into a fetus and a trophectoderm that can differentiate into a placenta It means fertilized egg that has developed enough to be distinguished.

In vitro fertilization (embryo) implantation requires an implant-related protein enzyme present in the cell membrane of the blastocyst, which is a cell membrane of the blastocyst. Since the normal in vitro fertilization embryo stops the premature division of the embryo and conception becomes difficult, Securing the trophoblast cells allows the uterine implantation to be induced more easily.

The present invention relates to a method for in vitro fertilization of a mammalian egg and sperm comprising the step of adding and correcting sperm and egg of a mammal to said in vitro fertilization medium composition.

The in vitro fertilization method of the present invention is a method for in vitro fertilization of a mammalian egg and sperm fertilization composition containing dipstick tosudan hydride 4 as an active ingredient by adding a sperm and an egg of a mammal to the fertilized egg, By increasing the abandon development rate, embryo transfer can be facilitated.

(A) obtaining follicles from a mammal other than a human;

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

(c) in vitro maturation of the immature oocytes recovered in step (b);

(d) in vitro fertilization of oocytes and spermatozoa matured in step (c) by adding to the medium composition for in vitro fertilization of mammalian egg and sperm containing Diffractosidanhydride 4 as an active ingredient; And

(e) culturing the in vitro fertilized egg modified in the step (d) in a culture medium.

The present invention also provides a diluted liquid semen diluted with a liquid comprising 0.05 to 0.5% (w / v) of Difructose dianhydride 4 as an active ingredient.

Preferably, the dilution of the pig liquid semen dilute comprises 0.05 to 0.75% (w / v) of dipstick tosudanhydride 4, more preferably 0.05 to 0.5% (w / v) Is 0.15% (w / v), and most preferably 0.1% (w / v), but is not limited thereto.

The diluted pig liquor is preferably a BTS (Beltsville thawing solution) dilution (3.7 g glucose, 0.6 g sodium citrate, 0.125 g sodium bicarbonate, 0.125 g 0.0 &gt; g., &Lt; / RTI &gt; penicillin G, 0.1 g streptomycin) may contain dipstixedanhydride 4 in the above amounts, But is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited thereto.

Example  1. Preservation of motility of pig sperm

To determine the effect of Diffractosidian hydride 4 on sperm motility retention, pig semen samples from pig farms were analyzed for motility (> 85% viability) and sperm concentration, followed by a Beltsville thawing solution (100 ml distilled water (BTS dilution) containing 1 x 10 ⁸ sperm counts / ml, containing 3.7 g of glucose, 0.6 g of sodium citrate, 0.125 g of sodium bicarbonate, 0.125 g of Ethylenediaminetetraacetic acid, 0.075 g of potassium chloride, 0.06 g of penicillin G and 0.1 g of streptomycin) And stored at 17 ° C for 5 days. At this time, different concentrations of dipstick tosudanhydride 4 (0.1, 0.25, 0.5, 0.75%) were added to the BTS diluent and the control was spermatozoa in BTS without Diffractosidanhydride 4.

Then, the motility of the sperm was observed with an optical microscope equipped with a 37.5 ° C heating plate.

As shown in Fig. 1, sperm motility did not show a significant difference among the treatment groups for 4 days after liquid semen storage, but showed high motility in the group to which 0.1% diproxysthidone hydride 4 was added. On the fifth day of storage, the sperm motility was significantly higher in the group to which 0.1% diproxothiadiene hydrate 4 was added (77% mobility) (0 or 0.25-0.75% -71% mobility, p <0.05; Fig. 1). This suggests that addition of 0.1% diproxysthidone hydrate 4 is effective in preserving the motility of porcine sperm.

Example  2. Of pig sperm Active oxygen species ( ROS ) Occurrence rate

Activated oxygen species (ROS) from pig spermatozoa cause peroxidation, DNA damage, and fertilization of spermatozoa, but appropriate levels of reactive oxygen species (ROS) are involved in sperm motility and fertility.

0.1%, 0.25%, 0.5% and 0.75% (w / w) of dipstick tosudan hydride 4 on the production of reactive oxygen species (ROS) in spermatozoa (RTS) at 10, 30, and 60 minutes after diluting the semen with 1 × 10 ⁸ sperm / ml in a pig semen dilution (Beltsville thawing slout, BTS) Was confirmed by fluorescence intensity.

As a result, it was confirmed that the production of reactive oxygen species was reduced in the porcine seminal diluent added with the concentration of 0.1-0.5% (w / v) of Diffractosidone hydride 4 (FIG. 2). This indicates that Diffractosidone hydride 4 can regulate the level of reactive oxygen species generated during sperm culture.

Example  3. Production of porcine in vitro fertilization medium

The medium for in vitro fertilization contained 0.1 to 0.75% (w / v) of Diffractosidianhydride 4 (DFA 4), and mTBM supplemented with 0.2% bovine serum albumin (BSA) The medium for in vitro fertilization was prepared in a Tris-buffered medium containing 113.1 mM NaCl, 3.0 mM KCl, 7.5 mM CaCl ₂ .2H ₂ O, 20.0 mM Tris, 11.0 mM glucose, 5.0 mM sodium pyruvate and 3.4 mM caffeine.

Example  4. In vitro maturation of pigs Follicular  Ready

The ovaries were slaughtered at the slaughterhouse and the ovaries were stored in a physiological saline solution at 25 ° C and stored at 25 ° C in a physiological saline solution. The ovaries were transferred from the follicles 2 to 6 mm in diameter using a syringe equipped with an 18- Immature oocytes with follicular fluids and cumulus cells were inhaled.

The obtained immature oocytes were diluted in a culture medium of TL-Hepes (Voelkel, SA and Hue, Y, Theriogenology 37, 1117-1131, 1992) containing 0.01% (w / v) polyvinyl alcohol Healthy immature follicles were selected. The criteria for selection is an immature ovarian follicle in which cytoplasm is uniformly distributed and three or more layers of follicular cells are closely attached.

The immature oocytes of the selected pigs were treated with 0.1% PVA, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 0.5 μg / ml lutenizing hormone (LH), 0.5 μg / ml follicle (GIBCO BRL, Cat. No. 31100-35) supplemented with 10 ng / ml epidermal growth factor (EGF), 75 / / ml penicillin G and 50 / / ml streptomycin ), And then in vitro maturation was induced by culturing in a 5% CO ₂ incubator at 38.5 ° C for 44 hours in an incubator.

Example  5. Investigation of porcine oocyte binding count of pig sperm

In Example 4, oocyte cells inoculated with oocytes were removed using 0.1% (v / v) hyaluronidase in vitro for 44 hours to obtain a first polar body, And only the egg of metaphase II is protruded.

DFA 4 was added to the in vitro fertilized medium supplemented with 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% (w / v) Selected oocytes (10 eggs per 100 μl mTBM) were added and prepared. As a control, mTBM medium containing 0.2% bovine serum albumin without DFA 4 was used.

1 ml of the liquid semen of the pig stored at 17 ° C was mixed with 5 ml of PBS containing 0.1% (w / v) polyvinyl alcohol (PVA) and centrifuged at 2,000 rpm for 5 minutes. Respectively.

After centrifugation, only the immersed spermatozoa were recovered and diluted with mTBM medium. DFA 4 was added to each of 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% ) Was added at a concentration of 5.0 × 10 ⁵ sperm / ml and the in vitro fertilization was induced in a 5% CO ₂ incubator at 38.5 ° C. for 1 hour. Thereafter, the number of spermatozoa bound to porcine oocytes was confirmed.

As a result. The binding of DFA 4 to oocytes in porcine spermatozoa was increased in vitro fertilization medium supplemented with 0.1% (w / v) and 0.25% (w / v) concentrations (FIG. 3).

Example  6. Production of pig embryos

In Example 4, oocytes that have been oocyte-encapsulated in oocytes matured for 44 hours were removed with 0.1% (v / v) hyaluronidase and the cytoplasm was healthy and the first polar body Only the protruding metaphase II eggs are selected.

DFA 4 was added to the in vitro fertilized medium supplemented with 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% (w / v) Selected oocytes (15-20 oocytes per 100 μl mTBM) were added and prepared. As a control, mTBM medium containing 0.2% bovine serum albumin without DFA 4 was used.

1 ml of the liquid semen of the pig stored at 17 ° C was mixed with 5 ml of PBS containing 0.1% (w / v) polyvinyl alcohol (PVA) and centrifuged at 2,000 rpm for 5 minutes. Respectively.

After centrifugation, only the immersed spermatozoa were recovered and diluted with mTBM medium. The DFA 4 containing the eggs was added to the medium containing 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) (w / v) was added to the in vitro fertilization medium at a concentration of 1.0 × 10 ⁵ sperm / ml, and the in vitro fertilization was induced in a 5% CO ₂ incubator at 38.5 ° C. for 2 hours.

Example 7:  Incidence rate of embryos

In vitro fertilization medium supplemented with 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% (w / v) In vitro fertilization-induced in vitro fertilization was carried out in a PZM-3 medium (Yoshioka et al., Biology of reproduction 66 (1): 112-119, 2002) at 38.5 ° C in a 5% CO ₂ incubator After culturing for 20 hours, the cultured embryos were fixed with 2% (v / v) formaldehyde and stained with 2.5 mg / ml DAPI (4 ', 6-diamidino-2-phenylindol) , And normal fertilization rates of embryos were examined using fluorescence microscopy.

A monospermic oocyte was detected when one of the magnetic and progenitor nuclei were observed in the embryo, and a polyspermic oocyte was detected when more than one male nucleus was observed. A high percentage of polyspermy slows or prevents the development of normal embryos after fertilization.

According to the Example 3, DFA 4 was added to the in vitro fertilized medium supplemented with 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% The normal fertilization rate of the fertilized eggs was 59.8 ± 4.3% in the fertilized medium supplemented with 0.1% (w / v) of DFA 4 and 0.25% (w / v) in the control and DFA 4, The normal fertilization rate of the IVF medium supplemented with 0.5% (w / v) and 0.75% (w / v) was 43.8 ± 6.1 to 51.0 ± 10.2% (FIG.

Thus, the fertilized egg fertilization rate of fertilized embryos in the IVF medium supplemented with 0.1% (w / v) DFA 4 was 0.25% (w / v), 0.5% (w / v) and 0.75% w / v) compared to the normal fertilization rate of the in vitro fertilization medium.

It was confirmed that the in vitro fertilization medium supplemented with 0.1% DFA 4 had the effect of promoting the normal development of porcine IVF embryos.

Example  8. Pigs As a blastocyst  Development rate survey

In vitro fertilization medium supplemented with 0.1% (w / v), 0.25% (w / v), 0.5% (w / v) and 0.75% (w / v) In vitro fertilization-induced in vitro fertilization was carried out in a PZM-3 medium (Yoshioka et al., Biology of reproduction 66 (1): 112-119, 2002) at 38.5 ° C in a 5% CO ₂ incubator After 6 days of incubation, embryo development rate and blastocyst development rate of in vitro fertilized embryos were confirmed.

As a result, the fertilization rate of fertilized eggs modified with the DFA 4 supplemented with 0.1% (w / v) was 80.0 ± 3.5%, and the control and DFA 4 were 0.25% (w / v) and 0.5% w / v) and 0.75% (w / v), the fertilization rate of fertilized eggs was 68.8 ± 1.3 ~ 78.8 ± 5.1%.

In vitro fertilization medium supplemented with 0.1% (w / v) DFA 4, the blastocyst formation rate of fertilized embryos was 36.8 ± 4.6% and the control and DFA 4 were 0.25% (w / v) and 0.5% (w / v) ) And 0.75% (w / v) of embryos were 10.9 ± 2.5 ~ 25.4 ± 3.6% in the fertilized embryos and the number of cells per bladder was 0.1% (w / v) ) And the control and DFA 4 were added to the in vitro fertilization medium supplemented with 0.25% (w / v), 0.5% (w / v) and 0.75% (w / v) And 33.3 ± 3.1 ~ 40.4 ± 2.6, respectively.

Therefore, in vitro fertilization medium supplemented with 0.1% (w / v) of DFA 4, the fertilization rate and blastocyst formation rate of the fertilized eggs were 0.25% (w / v) and 0.5% (w / v) And 0.75% (w / v) of fertilized embryos were higher than those of fertilized embryos and blastocyst formation rate (FIG. 5).

In vitro fertilization medium supplemented with 0.1% (w / v) DFA 4, the average number of cells per blastocyst was 0.25% (w / v), 0.5% (w / v) and 0.75% w / v) (Fig. 6).

It was confirmed that the in vitro fertilization medium supplemented with 0.1% of DFA 4 had the effect of improving the embryo development of pig embryos.

Claims (7)

Which enhances the normal fertilization and embryo development rate of in vitro fertilized oocytes containing 0.05 to 0.15% (w / v) of Difructose dianhydride 4 as an active ingredient, A medium composition for in vitro fertilization. The culture medium for in vitro fertilization of pigs and spermatozoa according to claim 1, wherein the culture medium composition further comprises at least one selected from inorganic salts, carbon sources, amino acids and bovine serum albumin. delete delete delete (a) collecting follicles from the pig;
(b) recovering immature oocytes from the oocytes collected in step (a);
(c) in vitro maturation of the immature oocytes recovered in step (b);
(d) in vitro fertilization of oocytes and spermatozoa matured in step (c) by adding the oocytes and spermatozoa to the in vitro fertilization medium composition of claim 1 or 2; And
(e) culturing the in vitro fertilized egg modified in step (d) in a culture medium. delete

Images