KR101390536B1

KR101390536B1 - Mass producing method for high meat quality korean native cattle by in vitro fertilization technique

Info

Publication number: KR101390536B1
Application number: KR1020130033861A
Authority: KR
Inventors: 김민규; 박연배; 한길우; 이준헌; 이경본; 장옥; 이기곤; 정일윤; 이성구
Original assignee: 전라북도 임실군(임실군 농업기술센터장); 충남대학교산학협력단
Priority date: 2013-03-28
Filing date: 2013-03-28
Publication date: 2014-05-07

Abstract

The present invention relates to a method for securing and preserving a superior genetic resource of Korean beef cattle by collecting oocytes in vivo by selecting a high-capacity fertilizer having excellent genetic ability and mass-propagating excellent embryos by biotechnology and embryo transfer technology, Furthermore, it provides models and techniques for securing the competitiveness of differentiated Hanwoo for increased income and imported meat by keeping the high quality meat production system.

Description

[0002] The present invention relates to a method for mass production of high-performance Korean cattle using embryo transfer technology,

The present invention relates to a method for producing high-yield Korean milk beef to secure economic efficiency by mass-propagating high-yield Korean beef using biotechnology and embryo transfer technology, to secure, preserve and maintain a superior genetic source of Korean beef, .

Since the price of Hanwoo is mainly based on the meat quality grade and the quality of the meat is known to be 60% or more by inheritance, securing the gene of good Hanwoo and producing good Hanwoo is the best shortcut to securing the economical efficiency of the farm. It is also essential to deliver good traits to the later generations.

However, due to the lack of a systematic embryo transfer program in Korea, the embryo transfer efficiency is significantly lower than that of foreign countries, and the quality of Hanwoo production system is mainly hydrogen. In other words, the production of high-performance Hanwoo is mainly the result of verifying the ability of the seedling, ie, the bull, and the hereditary ability is evaluated. In addition, the number of cows with dominant traits and pedigrees is low as well as the number of domestic cows is very low. In addition, the method of collecting the eggs from the slaughterhouse has a problem that the quality of the eggs is lowered and the fertilization rate is lowered, so that the fertilization rate is lowered and the fertility rate is lowered.

Korean Patent No. 10-1064313

The present invention has been made to overcome the problems of the prior art as described above, and it is an object of the present invention to provide a method for selecting a Korean beef cattle having excellent traits and feeding them to a livestock farm, Determining the presence or absence of a specific SNP for screening, obtaining an ovum from an ovum pick-up selected from an OPU (Ovum Pick Up) using an ultrasonic diagnostic apparatus, a step of in vitro fertilization of the spermatozoa from the spermatozoa, Culturing an in vitro fertilized embryo to form a blastocyst, and introducing the blastocyst into an estrus synchronized feeder.

In order to accomplish the object of the present invention as described above, the present invention provides a method for identifying SNPs, comprising the steps of: Collecting oocytes from a well-selected empty ovum selected by OPU (Ovum Pick Up) using an ultrasonic diagnostic apparatus; A step of in vitro fertilization of the collected oocyte with a sperm-derived sperm; Culturing the in vitro fertilized embryo to form a blastocyst; And introducing the blastocyst into an estrogen-synchronized feeder.

In one embodiment of the present invention, the presence or absence of the SNP may be confirmed by a PCR (Polymerase Chain Reaction) -RFLP (Restriction Fragment Length Polymorphism) method or a PCR product sequencing method.

In one embodiment of the present invention, the collected oocytes can be matured for 12 to 32 hours using an incubator supplied with 3 to 7% (v / v) CO ₂ .

In one embodiment of the present invention, the step of culturing the embryo can be cultured in a culture medium supplemented with a plasminogen activator.

In one embodiment of the present invention, the plasminogen activator may be FGF10.

In one embodiment of the present invention, estrogen synchronization may be induced by treating the hormone into the recipient from 8 to 10 days prior to the introduction of the blastocyst.

In one embodiment of the present invention, the hormone may be any one selected from PGF2alpha (PG: prostaglandin F2), GnRH (Gonadotropin Realeasing Hormone), PMSG and estrogen.

Further, the present invention provides a high-quality cow produced by the method of the present invention.

The present invention is based on the first technical aspect of the present invention for the development and establishment of oocyte retrieval technology without induction of follicular development. It has been established that embryo production technology using the superior oocyte produced in the body has been developed, Mass production is possible.

In addition, to save the cost of production of high-performance Hanwoo embryos, we select individuals with high quality genetic traits and maximize the conception rate by producing the best fertilized eggs by establishing the best in vitro fertilization method.

It is also possible to obtain the best conception rate by measuring the condition of the paddyweed, and to form a new meat quality excellent Hanwoo axis species and to improve the cow improvement effect.

Secondly, in terms of economic and industrial aspects, farm income is improved by increasing the conception rate (over 50%) in order to establish a technology for obtaining superior gene-containing oocytes by low-cost and low- do.

In addition, breeding and mass production of Hanwoo with excellent gene can be improved through the transplantation of embryos. Also, it is possible to strengthen the competitiveness and industrialization of Hanwoo farmers by establishing the improvement system of high-performance Hanwoo. It can contribute to securing excellent Hanwoo genetic resources and increasing competitiveness of farm households that directly generate high income for farmers.

Figure 1 shows the SNP of nucleotide 171 of CAPN1-316 gene.
Figure 2 shows the SNP of the CAPN1-4751 gene 62 nucleotide.
Fig. 3 shows the SNP of nucleotide No. 68 of the CAPN3 gene.
Fig. 4 shows the SNP of the 49th nucleotide of the CAST gene.
5 shows the SNP of nucleotide No. 86 of the CAST gene.
FIG. 6 shows the result of RFLP (restriction fragment length polymorphism) of the FASN gene.
7 is a view showing an ultrasound image screen.
FIG. 8 shows the average value of the results of performing the monthly OPU from the five blank lines.
FIG. 9 shows an average value of the number of oocyte retrieval by number in each blank.
FIG. 10 shows an in vitro fertilized state.
Fig. 11 shows a photograph of staining a blastocyst.

In the present invention, advanced genetic technology (DNA marker) was applied to improve utilization and efficiency of embryo transfer technology for efficient and continuous improvement of Hanwoo and farm income.

In the case of advanced livestock, DNA markers that can be put to practical use over the past decade have been continuously explored to discover genes related to meat and meat quality, and these technologies can be used to systematically improve embryo transfer technology. In the present invention, the attempt was made to estimate the genetic parameter, but the related farmers could not cooperate with each other, and a blank line was selected using an advanced method using a DNA marker. In this study, we investigated the genes for CAPN1-316, CAPN1-4751, CAPN1-4751, CAPN3, CAST and oil FASN related to oleic acid, which is unsaturated fatty acid, in Korean cattle through a joint study with the Genetics Laboratory of Chungnam National University.

In the present invention, high-quality Korean beef refers to a cow produced with excellent qualities for high quality meat under the systematic specification control of hydrogen, and in the case of cow, it is supplied to the bottom of a farmhouse to achieve uniform production quality.

OPU (Ovum Pick-up) is an advanced technology that directly extracts eggs by using ultrasound equipment from ovaries of living cows as an ovum extraction technique. The difference between the OPU method and the conventional method of producing embryos is that the hormone is not used in the ovum for ovum retrieval, thus securing the health of the cow and the safety of the reproductive organs. Second, it can produce more than 50 calves for 4 months.

Oestrus Synchronization means that the Hanwoo becomes mature and the average is 21 days when it is not pregnant. Therefore, the breeding farm must observe the daily estrus, and when the estrous cycle is missed, additional breeding stock is required. In particular, while the quality of feed and quality of control are improving, the amount of exercise is insufficient and body fat accumulation is caused excessively, so that the number of individuals with mild fever is increased and the estrus of the herd is not easy to observe. Therefore, the estrogen synchronization method artificially induces the development and maturation of the follicles, or regresses the luteal phase, and the other method is to prolong the survival of the luteal to allow the estrus and ovulation at a certain time. In the meantime, the estrogenic hormone agent that has been widely used can be classified into two types according to its purpose. Progesterone or a progesterone analogue similar to the progesterone analogues thereof inhibits the development and maturation of the follicles. The regenerating progesterone includes PGF2 Or a derivative thereof is used.

The present invention is characterized in that it provides a method for selecting and preserving high quality Korean beef and for improving and preserving the genetic resources of high quality Korean beef cattle after transplanting after fertilization and sperm retrieval.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to 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 to these embodiments.

< Experimental Example >

After screening three farms among Hanwoo farmers who wanted to do business, about 32 candidates were selected and their genetic ability was verified by blood sampling. Based on the results of analysis using SNP markers that determine the meat quality and taste of Hanwoo, five genetic tests were selected and five commercial samples were purchased according to the purchasing procedure. It is transported to the Animal Resource Research Center, and it is being harvested by thoroughly managing the specimens and breeding the blank.

OPU-related ultrasound equipment was purchased on December 02, 2010, and fertilized eggs were produced twice a week at the Animal Resource Research Center of Chungnam National University. Production efficiency of fertilized eggs was increased by multiplying fertilized egg production -40), respectively, which were significantly higher than the efficiency (120-150 per year).

Spermatozoa were purchased from the Nonghyup Hanwoo Rehabilitation Center, and spermatozoa with high production efficiency were selected. Spermatozoa were used to exchange an individual for a month to prevent ancestry.

The embryos were transferred to a portable incubation incubator in order to shorten the embryo transfer time and to reduce the exposure time of the embryos. The embryo transfer was performed within 3 hours, And the embryo transfer was performed 10 days before embryo transfer through estrus synchronization and intrauterine environment analysis.

The embryo transplantation was performed by judging the ovarian status and estrus phenomenon clinically. The abnormal embryos were excluded from the embryo transfer, and the pregnancy diagnosis was decided to be performed collectively by petition of the farmer. .

< Example 1>

rainfall Blank selection

<1-1> candidates Blank Selection and Blood Collection

Three farm households were selected from the Hanwoo farms in Imsil County and the candidates were selected based on their health status, pregnancy status, age, living quarters, and nutritional status. In order to confirm the genetic capability, 32 blood samples were collected from the blood samples of five Korean beef cattle.

<1-2> candidates Blank Genetic Marker Confirm

FIGS. 1 to 5 show the results of using the markers for CAPN1-316, CAPN1-4751, CAPN3, and CAST genes.

FIG. 6 shows RFLP test results to confirm the gene FASN associated with oleic acid, which is an unsaturated fatty acid.

Selection markers of traits related to meat quality No Sample ID CAPN1-316 CAPN1-4751 CAPN3 CAST FASN pasture
Lord SNP g.5679 C> G SNP g.6545 C> T SNP g.46415 G> T SNP g.134897 T> C SNP g.17924 G> A CC CG GG CC CT TT GG GT TT TT TC CC GG GA AA 12 8869 X X X X X Kim xx 17 7414 X X X X X 00 18 7111 X X X X X New yy 27 3098 X X X X X Kim xx 30 8260 X X X X X Kim xx % 66.70% 20% 13.30% 43.30% 46.70% 10.00% 73.30% 23.30% 3.40%

1. SNP g.5709 C> G of CAPN1-316 gene containing G allele was associated with increased shear force and C was less associated with shear force. The SNP was different with our SNP (g.5679 C> G).

2. SNP g.6545 C> T of CAPN1-4751 gene was associated more terder in CC & CT genotype compare to TT genotype.

3. CCT genotype was more though than TT genotype.

4. SNP of CAPN3 gene having GG genotype increased increased tenderness than other genotype.

5. CAST 5) SNP position is same with SNP patent (Barendse et al., 2002).

Calpain (CAPN) is a neutral cysteine protein hydrolyzing enzyme in animal cells. It acts as the first enzyme in postmortem tenderization, reducing Warner Bratzler Shear Force scores, and is known as a gene that affects the year of meat. It is known that it plays an important role in decomposing the protein of the human body and softening the meat quality.

Calpastatin (CAST) is a protein that specifically inhibits calpain with a molecular weight of 72,000 to 75,000. It forms a complex with calpain only in the presence of calcium and does not bind directly to the active center of calpain but covers the active center region It inhibits. The molecule has four repeats of about 152 residues, each of which can inhibit one molecule of calpain. The minimum inhibitory unit is about 30 amino acids. Most of them are known to be involved in the cytoplasm, some in the membrane, and affecting the fleshy year.

FASN gene was known as a result of genetic improvement of meat and meat quality of Hanwoo. Of the SNPs found in exon 39, GG type individuals showed higher levels of unsaturated fatty acids than AG type or AA type . This is a gene that increases the unsaturated fatty acid, and the more the GG type is, the thinner the backfat thickness and the larger the suture cross section.

SNP location and primer set Primer Set for Calpain and Calpastatin , SNP location and genotyping (based on sequencing result)

Gene Name / gene marker name GenBank Primer set Location PCR product size SNP
location Genotyping (Sequencing) Restriction enzyme (bp) References Acc. No. CAPN1 / CAPN1-316 AF 252504 F: 5'-GAGCTGGCCCTCATAAGATAA-3 ' Exon 9
220 bp
g.5679 C> G
CC = 20 (66.7%) BceAI Page et al., 2002 & GG: 124, 65 & 31 bp CG = 6 (20.0%) R: 5'-CCCATCCTCCATCTTGACC-3 ' GC: 155,124,65 & 31 GG = 4 (13.3%) CC: 155 & 65 bp CAPN1 / CAPN1-4751 AF
248054
F: 5'-AAGGGACAGATGTGGACAGG-3 ' Intron 17
144 bp
g.6545 C> T
CC = 13 (43.3%) BsaJI White et al., 2005
CC: 80, 62 CT = 14 (46.7%) Chromosome 29 R: 5'-GAGGGGTGTTCTCTGAGTGC-3 ' CT: 142, 80 & 62 TT = 3 (10.0%)
TT: 142 CAPN3 NC_007308 F: 5'-ATTGCATGGCCTCCTGAC-3 ' Intron 12
(46347-46573) 226 bp g.46415 G> T GG = 22 (73.3%) No restriction enzyme Barendse et al., 2008 GT = 7 (23.3%) (Chromosome 10) R: 5'-CTCCAGAACACCTCTGGACTG-3 ' TT = 1 (3.3%) CAST NC_007305 F: 5'-CCTCACGTGTTCTTCAGTGT-3 ' Exon 32
134414 ... > 136458 150 bp g.134869A> G
g.134897 T> C TT = 12 No restriction enzyme Barendse et al., 2002
(Patent) (Chromosome 7) R: 5'-ACGATTAGCAGCTCAAGAGGA-3 ' CC = 3 FASN
(Chromosome 19) AC_000176 F: 5'-TCTTCACAGAGCTGACGGAC-3 ' Exon 39 g.134869A> G AA = 27 DdeI AA: 46, 104 F: 5'-GGAGGAAGAGCTGTTGCAGT-3 ' AG = 2 AG: 46, 104, 150 GG: 150

<1-3> Candidates Blank Disease inspection and purchase

Results of Livestock Disease Test number Name of the celebration Vine species Place of collection Date of collection Inspection date Inspection number Brucella Tuberculosis Foot-and-mouth disease leukemia system No. 3 5 One 00 Hanwoo Farm 20101126 20101202 One Head
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voice 2 Kim xx Hanwoo Farm 20101126 20101202 3 Head
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voice 3 New yy Hanwoo Farm 20101126 20101202 One Head
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Results of individual animal disease test Serum number Name of the celebration Vine species gender Object number Brucella Tuberculosis Foot-and-mouth disease leukemia One 00 Hanwoo cancer 047074143 voice voice voice voice 2 Kim xx Hanwoo cancer 174288693 voice voice voice voice 3 Kim xx Hanwoo cancer 188682609 voice voice voice voice 4 Kim xx Hanwoo cancer 201730980 voice voice voice voice 5 New yy Hanwoo cancer 044971113 voice voice voice voice

3 infectious diseases selected by the farmers were examined by Jeonbuk Livestock Hygiene Laboratories and it was judged that they were not infected with brucellosis, tuberculosis, foot-and-mouth disease and leukemia.

< Example 2>

Blank Collection and modification

<2-1> Ultrasonic diagnostic equipment

Use and characteristics of ultrasonic diagnostic equipment A. Features (1) Utilizing OPU (oocyte retrieval system) system
(2) It is used to diagnose whether or not pregnancy diagnosis of sonar pig is early, and to diagnose whether there is disease of breeding period disease
(3) Utilizing ultrasound imaging data on specific parts of cattle and pigs for meat quality evaluation
(4) Easy to move and easy to use on site
(5) color Doppler ultrasound which can diagnose internal medicine and heart of various animals C. Usage (1) Production of blastocysts through the extraction of oocytes in bovine follicles and cultivation of high quality Korean beef
(2) Diagnosis of pregnancy after embryo transfer

By purchasing an ultrasound guide ovum harvesting device, it was possible to collect continuous ova from the wells.

<2-2> Collection of oocytes

The ovaries were removed and the OPU probes were inserted into the vaginal opening. The ovaries were removed from the ovaries by a local anesthesia using a 2% lidocaine.

After confirming ovarian follicle through the image of the ultrasonic diagnostic equipment, the egg was taken out using the pump and the process was immediately selected. The selected oocytes were transferred to 0.25 straw and transferred to the laboratory using the liver ovary transporter.

FIG. 7 shows an image of an ultrasound device image, which shows how an egg is guided and collected through a device.

<2-3> In vitro fertilization

The oocytes transferred to the laboratory were subjected to in vitro fertilization procedure and the oocytes were matured for about 22 hours using a 5% CO2 incubator. After freezing, high-performance high-performance Hanwoo sperm was thawed and fertilized eggs were spermatozoa.

After in vitro fertilization, 5% CO _{2 and} 5% O ₂ were incubated for about 7 days. High quality fertilized embryos from cultured embryos were used for screening transplants.

Figure 8 shows fertilized eggs modified through in vitro fertilization.

Developmental rate of blastocyst of OPU-derived oocytes in vitro Treatment Total no . oocytes Embryo development Cleavage (%) Morula (%) Blastocysts (%) control 165 125 (75.8%) ^a 64 (38.8%) 31 (18.8%) ^b OPU 162 99 (61.1%) ^b 55 (34.0%) 46 (28.4%) ^a

a-b Within a column, means without a common superscript differed, P <0.05. control (slaughterhouse-derived ovaries)

Table 6 above shows that the blastocyst formation rate of oocytes obtained from slaughterhouse is higher than blastocyst formation rate of oocytes obtained from slaughterhouse.

&Lt; Example 3 >

Conception Rate Program

<3-1> Estrous synchronization program

Synchronization methods of estrus worms have been the most conceivable method in years of experience, and induction of estrus using PGF2alpha and PMSG according to the estrus cycle of the wormworm has been carried out. To try to maximize the conception rate. As a result, estrus synchronization rate was 82%.

Synchronization rate of estrus Total Processing Head Estrus Synchronization% (%) Asynchronous head (%) 326 267 (81.9) 59 (18.1)

<3-2> Transplant Composition

In order to improve the transplantation efficiency, the plasminogen activator FGF10 was added to the transplantation medium to improve the transplantation efficiency.

<3-3> Suiranwoo Uterine environment Research

Experienced transplantation experienced veterinarians and veterinarians confirmed the condition of the uterus to determine the intrauterine inflammation, uterine hypertrophy, and uterine anomalies, and performed embryo transfer only in the cryopreserved uterus. Ovarian abnormality was observed in 5 of the total transplant recipients, and 3 cases of uterine inflammation were observed and treatment and culling were recommended.

<3-4> Excellent Blank Efficiency improvement program

In vitro fertilization (OPU) was carried out by selecting the bovine oocyte with the genes of good meat quality and tenderness, and the formation rate of embryo transferable blastocyst was higher than that of the normal oocyte from the slaughterhouse.

Embryo development after fertilization of oocytes obtained by OPU method Treated group Total experiment Number of eggs ship Development rate Split ratio (%) Loss of belly (%) Blastocyst (%) Control group 165 125 (75.8%) 64 (38.8%) 31 (18.8%) OPU 162 99 (61.1%) 55 (34.0%) 46 (28.4%)

The number of oocytes retrieved by OPU in the selected wells was found to be large when OPU was performed for 4-5 months on average, and it was 40 times or more twice a week This is in agreement with the phenomenon that the recovery rate of egg in the face is remarkably lowered.

FIG. 8 is a graph showing the average value of the results obtained by performing the monthly OPU from the five wells and FIG. 9 is the average value of the number of oocyte retrievals by the number of times in each well.

<Example 4>

Examination of fertilized eggs

<4-1> Morphological examination of embryos

The morphological evaluation of the embryos was judged to be satisfactory as a result of random selection of the first selection (self) embryos according to the classification standards of the International Association of Embryo Transfer (IETS) and the results were tested at the National Institute of Livestock Genomic Research Institute (Namwon).

<4-2> Cell number examination of blastocyst

The number of cells in the blastocysts formed to discriminate high quality blastocysts was very important, and randomized blastocysts were selected for cell counting. The test method was performed according to the international standard method. As a result, the number of cells in the blastocysts was judged to be 100 or more grade 1 embryos. Fig. 10 is a photograph of an embryo, and Fig. 11 is a photograph showing a staining of a blastocyst.

&Lt; Example 5 >

Transplantation and delivery

Local anesthesia was required because of environmental hormone changes during transplantation of the uterus. The estrus cycle was 21 days, and rutilization and PG were used as hormones for estrus synchronization. In the case of PG (prostaglandin F2) injection was injected into the uterine muscle.

It is a formulation containing natural prostaglandin PGF2a, which has a specific luteolytic action and a strong contraction function of the uterine smooth muscle, which can be applied not only in induction labor but also after delivery to dramatically improve uterine and reproductive diseases (PG), GnRH (Gonadotorpin Realeasing Hormone), PMSG and estrogen may be used depending on the estrus cycle of the water lily worm.

The ratio of hydrogen is slightly higher in cattle born from embryo transfer. It is advantageous to induce labor because the fetus and the receiver are dangerous after about 285 days.

Suwon Woo is a 5% higher conception rate than US Gyeongsan. For feed, it is recommended to feed hay and TMR feed for 3 ~ 4 months. Reducing the concentrate feed and raising the hay ratio to prevent the fattening will help to improve the conception rate.

Calves born from fertilized embryos due to the strong tendency of male sperm have a high proportion of hydrogen, but when using sperm separation machine, only female sperm can increase the proportion of cows. In the case of vaccination, completing 2 weeks before transplantation does not affect the transplant.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Attach an electronic file to a sequence list

Claims

Confirming the presence or absence of SNPs of any one of the five SNPs listed in the following table to select a good-quality blank or a seedling;
Collecting oocytes from a well-selected empty ovum selected by OPU (Ovum Pick Up) using an ultrasonic diagnostic apparatus;
A step of in vitro fertilization of the collected oocyte with a sperm-derived sperm;
Culturing the in vitro fertilized embryo with a culture medium to which a plasminogen activator is added to form a blastocyst; And
And introducing the blastocyst into an estrus-synchronized feeder.

The method according to claim 1,
Wherein the presence of the SNP is confirmed by PCR-RFLP method or PCR product sequencing.

The method according to claim 1,
Wherein the harvested oocytes are cultured for 12 to 32 hours using an incubator supplied with 3 to 7% (v / v) CO ₂ .

delete

The method according to claim 1,
Wherein the plasminogen activator FGF10 is the plasminogen activator.

The method according to claim 1,
Wherein the step of collecting the oocytes comprises collecting oocytes one to three times a week in a natural state from the high-quality empty oocyte

delete

A high-quality Korean beef produced by the method of any one of claims 1 to 3, 5 or 6.