KR101256581B1 - Method for transferring embryo in cow using solution for embryo transfer comprising topical anti-inflammatory agent - Google Patents

Abstract

PURPOSE: A method for transferring a fertilized egg of cows is provided to improve pregnancy rate in cows by suppressing an inflammatory reaction with dexamethasone and rhLIF, thereby increasing productivity of Korean beef cattle. CONSTITUTION: A method for transferring a fertilized egg of cows comprises the following steps: (a) externally aging a cow egg, externally fertilizing the egg with sperm, and externally culturing the fertilized egg; (b) putting the fertilized egg in a transferring solution containing dexamethasone and a recombinant human leukemia inhibitory factor(rhLIF), and loading the fertilized egg and the solution in straw; and (c) directly injecting the straw loaded with the fertilized egg and the solution, and transferring the fertilized egg. [Reference numerals] (1) Verifying based on the occurrence of re-estrus between 23 days and 42 days; (2) Analyzing pregnancy when 60 days after the pregnancy; (AA) Solution for transferring a fertilized egg; (BB) Basic solution(TCM-199 + 10% FBS); (CC) 100nM/ml Dexamethasone and 100nM/ml rhLIF; (DD) Washing twice to three times; (EE) Washing using the same solution; (FF) Loading a straw; (GG) Loading a fertilized egg and the solution for transferring a fertilized egg in the straw; (HH) Transferring the fertilized egg; (II) Analyzing pregnancy;

Description

Method for transferring embryo in cow using solution for embryo transfer comprising topical anti-inflammatory agent

The present invention relates to a method of implanting a fertilized egg of a cow characterized in that the implanted embryo implanted directly into the uterus of the cow with a fertilized egg transplant solution containing a topical anti-inflammatory agent.

In general, fertilized egg transplantation of cattle, horses, pigs, etc., refers to a fertilized egg obtained from an uterus of a cancer livestock before implantation, or by injecting a fertilized egg obtained by culturing an in vitro fertilized egg into a womb of a cancer livestock. . Such artificial fertilized egg transplantation can efficiently improve livestock improvement since it is performed by selecting a good livestock from among the same species of livestock. As an example of this, it is possible to produce high quality Korean beef from low-capacity cows using healthy cows as surrogate mothers.

The results reported to date indicate that the fertilized egg transplant industry has a lower efficiency in implantation rate, pregnancy rate and litter production efficiency than artificial insemination. This is judged to be due to various reproductive factors, among which stimulation of the uterus caused by fertilization during fertilization and implantation of the fertilized egg implantation machine causes inflammatory reactions. It has been reported that prostaglandin F2α (PGF2α) increases rapidly. In addition, it has been reported that the development, hatching, and implantation of blastocysts are reduced during transplantation of in vitro and ex vivo fertilized eggs due to an increase in PGF2α.

Therefore, a method of injecting an inflammatory inhibitor before fertilized egg implantation has been studied in order to lower the increase of PGF 2α caused by an inflammatory reaction in the uterus. For example, it has been reported to increase the pregnancy rate of livestock using flunixin meglumine, an existing anti-inflammatory agent used as a systemic injection.

However, the flunicin meglumine is a powerful anti-inflammatory agent that inhibits inflammation in the mother, but there is a risk of remaining in the mother, which affects breast milk, meat, etc., resulting in relatively low safety as food. In addition, the use of systemic injections such as anti-inflammatory drugs have been reported to affect breast milk and meat after pregnancy in humans and mice.

On the other hand, since topical anti-inflammatory drugs have a short duration, it is thought that the use of topical anti-inflammatory drugs instead of systemic anti-inflammatory drugs for fertilized egg implantation can only involve initial implantation and minimize inflammatory substances present in the uterus.

Therefore, in order to increase the low production efficiency caused by the inflammatory response generated during fertilized egg transplantation, when the fertilized egg is implanted directly into the uterus with a local anti-inflammatory agent, the fertilized egg transplanted by suppressing the inflammatory response caused by external factors It is thought that the implantation efficiency and pregnancy rate can be improved during implantation.

The present inventors have been studying to improve the low implantation efficiency and pregnancy rate caused by the inflammatory response during fertilization, and in vitro cultured fertilized eggs directly into the womb of a fertilized cow with a solution for transplanting fertilized eggs, including dexamethasone and rhLIF, which are PGF2α inhibitors. When implanted and transplanted, it was confirmed that the change in the amount of PGF2α in the plasma of the oviparous cow was little or decreased, and the pregnancy rate was improved compared to the cow transplanted using the fertilized egg transplant solution containing dexamethasone and rhLIF. It was.

Accordingly, the present invention is to provide a method for transplanting a fertilized egg of a cow characterized in that the implanted embryo is injected directly into the uterus of the cow together with a fertilized egg transplant solution containing dexamethasone and rhLIF PGF2α inhibitors.

The present invention

1) in vitro fertilization of bovine eggs, in vitro fertilized eggs and bovine sperm in vitro fertilization and in vitro culture,

2) in vitro cultured fertilized eggs in a solution for transplanting embryos containing dexamethasone and recombinant human leukemia inhibitory factor (rhLIF), and loaded into the straw, and

3) A method of transplanting a bovine fertilized egg, comprising the step of implanting a straw loaded with a fertilized egg and a fertilized egg transplant solution into the womb of a fertilized cow.

Hereinafter, the present invention will be described in detail.

The bovine fertilized egg transplant method according to the present invention is characterized in that the in vitro cultured fertilized egg implanted directly into the uterus of a fertilized cow with a solution for transplanting fertilized eggs, including dexamethasone and rhLIF PGF2α inhibitors.

The bovine fertilized egg transplant method of the present invention will be described in detail step by step.

Step 1) is a step of in vitro maturation of bovine egg, in vitro fertilized egg and bovine sperm in vitro fertilization and in vitro culture.

First, boil the eggs with D-PBS (Dulbecco's Phosphate-Buffered Saline) (containing 5.56 mM glucose, 1.25 mM sodium pyruvate, 15 μg / ml heparin sodium, 8 μg / ml phenol red and 10 μg / ml gentamicin). After washing three times, incubate IVMD101 (5.56 mM D-glucose, 0.91 mM sodium pyruvate, 5 mM taurine, 5 nM selenium, 5 µg / ml TGF, 10 ng / ml TGF) in 20 wells for each well in a 4 well multi dish. -α, 10 mg / ml Apo-transferrin, 1 mg / ml BSA, 5 mM HEPES and 10 μg / ml gentamicin), and incubators at 5% CO ₂ , 95% humidity and 39 ° C. conditions for 22-24 hours. Mature In Vitro

Next, wash the in vitro mature egg twice with IVF100 fertilization solution, transfer 30-40 eggs per 50 μl drop into IVF100 fertilization solution, and titrate the final concentration of sperm to 1.0 × 10 ⁷ / ml to obtain 50 μl IVF100. 50 μl of diluted semen is added to the fertilized solution and fertilized in vitro in an incubator at 5% CO ₂ , 95% humidity and 39 ° C. for 6 hours. The IVF100 correction solution is prepared by adding 1.25 mM sodium pyruvate, 0.5 mM cysteine, 5 mg / ml BSA, 7.5 µg / ml heparin sodium, 5 mM caffeine, and 10 µg / ml gentamicin to the BO correction liquid. Semen was thawed frozen Korean semen (KPN) for 30 seconds at 38 ℃, diluted in IVF100 fertilization solution and centrifuged for 5 minutes at 2,200rpm, and then recovered the highly active sperm precipitated in the lower layer and centrifuged again After washing with IVF100 correction solution, use.

In vitro fertilized eggs were then incubated with IVD101 culture (2.22mM D-glucose, 0.27mM sodium pyruvate, 2.48mM sodium lactate, 0.05mM L-cysteine, 200mM GSH, 5mM taurine, 5nM selenium, 10mg / ml Apo-transferrin, 10 ng / ml β-FGF, 1 ng / ml TGF-β1, 0.5 μg / ml TIMP-1, 0.5 μg / ml aprotinin, 1 mg / ml BSA, 5 mM HEPES and 10 μg / ml gentamicin) Culture in vitro at% CO ₂ , 5% O ₂ , 90% N ₂ , 39 ° C. In vitro cultures are changed every two days, and at the first replacement, the cells and sperm are removed from the cultures and incubated again until day 7 (early blastocyst).

Step 2) is a step of loading the embryos in vitro cultured fertilized eggs in solution for transplantation including dexamethasone and rhLIF. The fertilized egg transplant solution is prepared by adding 100 nM / ml dexamethasone and 100 nM / ml rhLIF to a TCM-199 solution containing 10% FBS (fetal bovine serum). The fertilized egg was washed 2-3 times with the fertilized egg transplant solution prepared above, and the fertilized egg and dexamethasone and rhLIF-added TCM-199 solution were loaded into 0.25 ml straw and transported at 37 ° C. to the fertilized egg transplant site. do.

In step 3) , the fertilized egg is implanted into the uterus of the fertilized cow. The straw loaded with the fertilized egg and the fertilized egg transplant solution is mounted on the catheter, and the fertilized egg is implanted into the uterus of the moored fertilized cow.

The cow transplanted by the above method had little or no change in the amount of PGF2α in the plasma of the cow after transplantation than before the embryo implantation, and the embryo that had been implanted using a fertilized egg transplant solution containing no dexamethasone and rhLIF. The pregnancy rate is improved. It is thought that inflammatory response is inhibited by PGF2α inhibitors dexamethasone and rhLIF, resulting in high pregnancy rate after fertilized egg transplant.

As described above, the method of transplanting bovine fertilized egg according to the present invention is a inflammatory reaction by dexamethasone and rhLIF by directly injecting the fertilized egg cultured in vitro with a fertilized egg transplant solution containing dexamethasone and rhLIF, which are PGF2α inhibitors, into the womb of a fertilized cow. This can be suppressed to improve the fertility rate of the eggs. Therefore, according to the method of transplanting the fertilized egg of the cow according to the present invention, not only can the production of Hanwoo be increased, but it is also possible to supply high quality improved Hanwoo, which can help to increase the income of farmers and improve the competitiveness of the livestock industry. do.

The method of transplanting bovine fertilized eggs according to the present invention has an effect of inhibiting the inflammatory response by PGF2α inhibitors dexamethasone and rhLIF, thereby improving the fertility rate of the eggs.

1 is a diagram briefly illustrating a process of implanting fertilized eggs into a fertilized cow using a solution for fertilized egg transplant containing dexamethasone and rhLIF.
Figure 2 is an in vitro model schematic diagram to overcome the inhibition of blastocyst hatching and implantation by PGF2α in the body during fertilized egg transplantation.
3 is a diagram showing the types of blastocysts cultured in vitro until day 9 to determine the blastocyst hatching rate of in vitro cultured fertilized eggs.
Figure 4 is a diagram showing the blastocyst development rate and hatching rate of fertilized eggs in vitro culture after addition of dexamethasone and rhLIF PGF2α inhibitor in vitro culture.
Figure 5 is a diagram observing the development rate and hatching rate of blastocyst of in vitro cultured fertilized egg after addition of dexamethasone and rhLIF PGF2α inhibitor in vitro culture.
Figure 6 shows the blastocyst development rate and hatching rate of fertilized eggs in vitro cultured after the addition of PGF2α inhibitor dexamethasone and rhLIF in the presence of PGF2α in vitro culture.
Figure 7 is a microscopic observation of blastocyst development and hatching rate of fertilized eggs in vitro cultured after the addition of PGF2α inhibitor dexamethasone and rhLIF in the presence of PGF2α in vitro culture.
8 is a diagram showing the change of PGF2α in the plasma of the oocytes before and after fertilization with the addition of PGF2α inhibitors dexamethasone and rhLIF during fertilization.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  One  Dexamethasone rhLIF How to implant a fertilized egg in the womb of a cow using a fertilized egg implant solution

One. Follicular  Recall and in vitro maturation

The ovaries of the slaughtered cows were removed from the slaughterhouse and washed three times with 0.9% saline at 15-20 ° C., and then placed in the same saline-filled thermos and transported to the laboratory within 2 hours. Next, eggs were collected from the visible follicles having a diameter of 2 to 7 mm using a 10 ml syringe with an 18 G needle. The collected oocytes were selected for in vitro maturation by selecting only eggs that had densely adhered to the cumulus cells and homogeneous cytoplasm under a stereoscopic microscope. The cultures used for washing and maturing the eggs were the methods reported in Yamashita et al. (1999). That is, the selected eggs were subjected to Dulbecco's Phosphate-Buffered Saline (D-PBS) (5.56 mM glucose, 1.25 mM sodium pyruvate, 15 μg / ml heparin sodium (sigma Chemical, St. Louis, MO), 8 μg / ml phenol red and In vitro maturation medium IVMD101 (5.56mM D-glucose, 0.91mM pyruvic acid) after washing 2 ~ 3 times with 10µg / ml gentamycin) and dividing 20-50 pieces per well into 4 well multi dishes (Nun, Roskilde, Denmark). Sodium, 5 mM taurine, 5 nM selenium, 5 μg / ml insulin, 10 ng / ml TGF-α, 10 mg / ml Apo-transferrin, 1 mg / ml BSA, 5 mM HEPES and 10 μg / ml gentamicin) Matured in vitro in an incubator at 5% CO ₂ , 95% humidity and 39 ° C. conditions for 24 hours.

2. In vitro fertilization

Eggs and sperm were treated using IVF100 fertilizer (Research Institute for the Functional Peptides, Yamagata, Japan). IVF100 correction fluid was added to BO correction fluid (Brackett and Oliphant, 1975) in 1.25 mM sodium pyruvate, 0.5 mM cysteine, 5 mg / ml BSA (Kokusai Shiyaku, Kobe, Japan), 7.5 µg / ml heparin sodium, 5 mM caffeine (Wako Pure Chemical Industries). , Ltd., Osaka, Japan), and 10 µg / ml gentamicin were added. Semen was used as frozen bovine semen (KPN) of Hanwoo guaranteed species, melted for 30 seconds at 38 ℃, diluted in the IVF100 fertilizer and centrifuged twice for 2 minutes at 2,200rpm, and then precipitated high activity sperm The cells were recovered, centrifuged again, and washed with IVF100 fertilizer. In vitro mature eggs were washed twice with IVF100 fertilization solution and 30-40 eggs per 50 μl drop were transferred to IVF100 fertilization solution.The final concentration of sperm was titrated to 1.0 × 10 ⁷ / ml and diluted in 50 μl IVF100 fertilization solution. 50 μl of semen was added and fertilized in vitro in an incubator at 5% CO ₂ , 95% humidity and 39 ° C. for 6 hours.

3. In vitro culture

In vitro fertilized eggs were cultured in IVD101 (2.22mM D-glucose, 0.27mM sodium pyruvate, 2.48mM sodium lactate, 0.05mM L-cysteine, 200mM GSH, 5mM taurine, 5nM selenium, 10mg / ml Apo-transferrin, 10ng / ml 5% CO ₂ using β-FGF, 1 ng / ml TGF-β1, 0.5 μg / ml TIMP-1, 0.5 μg / ml aprotinin, 1 mg / ml BSA, 5 mM HEPES and 10 μg / ml gentamicin) , 5% O ₂ , 90% N ₂ , in vitro culture at 39 ℃ conditions. In vitro cultures were exchanged every 2 days, and at the first replacement, the cells and sperm were removed from the culture medium and cultured again in vitro until day 7 (initial blastocyst).

4. Embryo Transfer

The in vitro cultured fertilized eggs were implanted into estrus-synchronized oviparous cows. Embryos were cultured from 0 to 7 days of fertilization using blastocysts, and 100 nM / ml dexamethasone and 100 nM / ml rhLIF were added to TCM-199 solution containing 10% FBS (fetal bovine serum). Embryos were washed 2-3 times with the same TCM-199 solution as above. Washed fertilized eggs and dexamethasone and rhLIF-added TCM-199 solution were loaded into 0.25 ml straw and transported at 37 ° C. to the fertilized egg transplant site. A straw loaded with fertilized eggs was mounted on the catheter and implanted into the womb of a mooring bovine cow.

A schematic diagram illustrating a process of transplanting fertilized eggs into a fertilized cow using a solution for fertilized egg transplant containing dexamethasone and rhLIF is shown in FIG. 1.

Comparative example  One  Dexamethasone rhLIF How to implant a fertilized egg in the womb of a cow using a fertilized egg transplant solution that does not contain

Except for fertilized egg in vitro fertilized in vitro in the same manner as Example 1, except that dexamethasone and rhLIF were not included in the fertilized egg transplant solution (TCM-199 solution) in Example 1 Transplanted.

Experimental Example  One  : Dexamethasone in vitro culture rhLIF Of in vitro cultured embryos after addition of Blastocyst Development rate  And hatching rate measurement

In order to determine the blastocyst development rate and hatching rate of in vitro cultured fertilized egg after addition of dexamethasone and rhLIF in vitro culture, the following experiment was performed.

Fertilized eggs produced through in vitro fertilization were in vitro cultured in IVD101 medium until day 5 (lost embryos), and after day 5, dexamethasone and rhLIF were added to CR2aa medium containing 0.15% BSA (no fatty acids) and 5% FBS (Sigma). 100 nM / ㎖ of each was added and cultured in vitro until the 9th day. After 9 days, in vitro cultured embryos were torn and the blastocysts in the escaped state were judged to be escaped blastocysts, expanded blastocysts without expansion, and expanded blastocysts and unexpanded blastocysts as initial blastocysts. And blastocyst development rate and hatching rate of in vitro cultured fertilized egg were observed under a microscope.

In vitro model schematic diagram for overcoming blastocyst hatching and implantation inhibition by PGF2α in fertilized embryos is shown in FIG. 2, and the types of blastocysts cultured in vitro until day 9 to determine blastocyst hatching rate in vitro cultured are shown in FIG. 3. Shown in In addition, measurement results and microscopic observations of blastocyst development and hatching rate of fertilized eggs in vitro cultured after addition of PGF2α inhibitors dexamethasone and rhLIF during in vitro culture were shown in FIGS. 4 and 5, respectively.

As shown in Figures 4 and 5, the addition of dexamethasone and rhLIF in vitro culture had no effect on the blastocyst development rate of in vitro cultured fertilized eggs, but the hatching rate showed high hatching rate when dexamethasone and rhLIF alone or in combination. Therefore, it can be seen that dexamethasone and rhLIF enhance the hatching rate of fertilized eggs in vitro.

Experimental Example  2  : In vitro culture PGF2 dexamethasone in the presence of α rhLIF Of in vitro cultured embryos after addition of Blastocyst Development rate  And hatching rate measurement

To confirm the blastocyst development and hatching rate of in vitro cultured fertilized eggs after addition of dexamethasone and rhLIF in the presence of PGF2α in vitro culture, the following experiment was performed.

Day 5 embryonic embryos produced in vitro were cultured in vitro to the 9th day of in vitro fertilization with dexamethasone and rhLIF with the addition of PGF2α, the hatching inhibitory factor. Then, in order to confirm whether the incubation also occurs in the secretion of PGF2α caused by inflammation as in the body under in vitro conditions, the blastocyst development rate and incubation rate of in vitro cultured fertilized eggs were measured and observed under a microscope.

Measurement results and microscopic observations of blastocyst development rate and hatching rate of fertilized eggs in vitro cultured after addition of PGF2α inhibitors dexamethasone and rhLIF in the presence of PGF2α in vitro culture were shown in FIGS. 6 and 7, respectively.

As shown in FIG. 6 and FIG. 7, when dexamethasone and rhLIF were added in the presence of PGF 2α in vitro culture, blastocyst development and hatching rate of in vitro cultured embryos were normal. Therefore, it can be seen that dexamethasone and rhLIF enhance the hatching rate of in vitro fertilized eggs. In addition, the improvement of hatching rate is due to the improvement of implantation rate and pregnancy rate is believed to increase production efficiency during fertilized egg transplantation.

Experimental Example  3  : Embryo Transfer PGF2 dexamethasone, an inhibitor rhLIF Before and after fertilization Suiranwoo  Present in plasma PGF2 measuring change in α

It has been reported that PGF2α, a product of inflammatory response in blood, is rapidly increased by an inflammatory response generated during fertilized egg transplantation. Therefore, blood was collected using venipuncture in the tail vein or artery of a fertilized cow (unpolished cow) 1 hour before and after transplantation of in vitro cultured embryos. The collected blood was placed in a heparinized tube and transported to a laboratory within 30 minutes to 4 hours using an ice bath. Blood samples were centrifuged at 1500xg for 10 minutes before plasma was separated. The separated plasma was stored at -20 ° C until analysis. The concentration of PGF2α in plasma samples was analyzed using an ELISA assay kit (CAY-516011, Cayman). The concentration of PGF 2α was calculated in pg / ml using the program provided by Cayman. The number of analyzed groups was 4 control groups (Comparative Example 1) and 13 treatment groups (Example 1), and the control group was implanted using fertilized egg transplant solution using dexamethasone, a PGF2α inhibitor, and rhLIF. , The treatment group is a group that was implanted using fertilized egg transplant solution containing dexamethasone and rhLIF.

The results are shown in Fig.

As shown in FIG. 8, in all four control groups, PGF2α increased rapidly in bovine plasma 1 hour after 1 hour before transplantation, and the inflammatory response by fertilized egg transplantation was observed. However, in 14 treatment groups treated with dexamethasone and rhLIF in parallel, it was confirmed that there was little or no change in the amount of PGF2α in the plasma of the oviparous cow 1 hour after 1 hour. These results are the same as the results presented by the in vitro model, and proved that the inflammatory inhibitors alleviate the rapid increase in PGF2α even in the body.

Experimental Example  4  : Embryo Transfer PGF2 dexamethasone, an inhibitor rhLIF Pregnancy Rate after Embryos Transplantation with Different Diets

In order to measure the fertility rate after fertilized egg transplantation with the addition of PGF2α inhibitors dexamethasone and rhLIF during fertilization, veterinarians were reestablished between 23 and 42 days after fertilized egg implantation and rectal examination and ultrasonography at 60 days. It was confirmed through. The control group (Comparative Example 1) was carried out with 75 dogs, the treatment (Example 1) was carried out with 126 dogs.

The results are shown in Table 1.

Transplant head Pregnancy head Pregnancy rate Control (Comparative Example 1) 75 42 56% Addition Sphere (Example 1) 126 86 68.3%

As shown in Table 1, the control group had a 56% implantation and pregnancy efficiency after fertilized egg transplantation, and the control group had a 68.3% implantation and pregnancy efficiency after fertilized egg transplantation. As a result, it can be seen that the fertility rate after fertilized egg transplantation of the oviparous cow of the present invention was increased to 12.3% compared to the control group, so that the inflammatory response was suppressed by PGF2α inhibitors dexamethasone and rhLIF, resulting in high fertility rate after fertilized egg transplantation.

Claims (4)

1) in vitro fertilization of bovine eggs, in vitro fertilized eggs and bovine sperm in vitro fertilization and in vitro culture,
2) in vitro cultured fertilized eggs in a solution for transplanting embryos containing dexamethasone and recombinant human leukemia inhibitory factor (rhLIF), and loaded into the straw, and
3) A method of transplanting a bovine fertilized egg, comprising the step of implanting a straw loaded with a fertilized egg and a solution for fertilized egg implantation directly into the womb of a fertilized cow. The fertilized egg transplant of claim 1, wherein the fertilized egg transplant solution is prepared by adding 100 nM / ml dexamethasone and 100 nM / ml rhLIF to a TCM-199 solution containing 10% FBS (fetal bovine serum). Way. The method of claim 1, wherein the fertilized egg is blastocyst fertilized egg cultured from 0 to 7 days when fertilized, bovine fertilized egg transplant method. According to claim 1, wherein the bovine sperm is frozen frozen semen (KPN) of Hanwoo boiled cattle for 30 seconds at 38 ℃, diluted in vitro fertilization solution and centrifuged to recover the highly active sperm precipitated in the lower layer, characterized in that Bovine fertilized egg transplant method.

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