KR101349009B1 - In vivo production method of bovine embyos by emporary undernutrition - Google Patents

Abstract

The present invention relates to a mammalian body fertilized egg production technology, and more particularly to a body fertilized egg production technology to induce over-ovulation using a transient low nutrient status of mammals. According to the present invention, it is possible to increase the number of implantable fertilized eggs for Hanwoo blank cows. Through this, it is possible to reduce the effort, processing cost, and feed cost according to the treatment of empty ovulation and recovery of fertilized eggs, and it is possible to improve high quality cow breeds with high quality grade.

Description

In vivo production method of bovine embyos by emporary undernutrition}

The present invention relates to a mammalian body fertilized egg production technology, and more particularly to a body fertilized egg production technology to induce over-ovulation using a transient low nutrient status of mammals.

Embryo transplantation, which has been put to practical use in cows, causes ovulation in cows with genetically superior abilities to fertilize a large number of eggs, and fertilizes them with excellent sperm semen. It is a technology that produces a large number of calf at a time by transplanting and fertilizing a baby, and is composed of overovulation, fertilization, fertilization and recovery of fertilized eggs, preservation of fertilized eggs, synchronous synchronization of blank and fertilized cows, and fertilized egg transplant technology. A cow is pregnant by ovulating one egg at a time, and produces approximately one calf per year. However, the eggs are harvested by over-ovulation technology and 5-10 fertilized eggs are collected at a time. Thus, 15-30 implantable embryos can be fertilized. In general, an average of one-time overovulation treatment in Korea yields 5.5 implantable embryos (6.1 overseas). The present invention aims at optimizing over-ovulation treatment technology and improving the efficiency of transplanting fertilized eggs from genetically superior Hanwoo cows, thereby reducing the cost of expensive fertilized egg recovery, preserving excellent genetic resources, and improving the efficiency of cow improvement. have.

The prior art of the present invention is the development research of a surrogate cow (pp.88) presented at the 3rd International Symposium and Conference of the Korean Society of Developmental Biology (2003).

Under these technical backgrounds, the present inventors have made intensive efforts to complete the present invention.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for producing an in vitro fertilized egg comprising inducing and ovulating superovulation in a transient low nutritional state of a mammal.

According to one aspect of the present invention, in vitro fertilization production method can be provided by inducing over-ovulation in a temporary low nutritional state of mammals.

According to one embodiment, the low nutritional state can be induced by low nutrition intake so that the total amount of plasticized nutrients (TDN) 1 ~ 2kg for the target mammal.

According to one embodiment, the low nutrition feeding period may be 20 to 25 days.

According to one embodiment, egg harvest may be made within 24 hours after the low nutrition feeding.

According to one embodiment, the mammal may be a cow.

According to the present invention, it is possible to increase the number of implantable fertilized eggs for Hanwoo blank cows. Through this, it is possible to reduce the effort, processing cost, and feed cost according to the empty ovulation over-ovulation treatment and fertilized egg recovery, and to improve the high-performance cow breed with high quality grade.

1 is a schematic diagram showing a superovulation treatment program for creating a temporary low nutritional state according to the present invention.

Hereinafter, the present invention will be described in more detail.

According to an aspect of the present invention, there may be provided a method for producing an in vivo fertilized egg of a mammal, including inducing and ovulating after inducing superovulation using a temporary low nutritional state.

According to one embodiment, the mammal may be one selected from the group consisting of cattle, pigs, dogs, cats and horses, preferably may be cattle.

The present invention is a technique for improving the production efficiency of implantable fertilized egg, that is, the improvement of over-ovulation and fertilization efficiency, which are common for the activation of fertilized egg transplantation technology for mammals, especially cattle.

In general, it is known that high quality fertilizers and nutrition are required to maintain optimal health status as well as educational textbooks. As a way to limit the amount of formula feed, that is, the temporary low-nutrition diet management that does not feed the formula, only the rice straw free vegetarian diet, the number of eggs and transplantable fertilized egg 10.9 more than double that of 5.5 Implantable fertilized eggs that can be harvested are related to production efficiency enhancing technology.

On the blank  Induction of low nutritional status

According to one embodiment, the low nutritional state may be induced by low-nutrition feeding so that the total amount of plasticized nutrients relative to the target mammal 1 to 2kg.

According to one embodiment, the low-nutrition state may be a low-nutrition intake so that the total amount of plasticizing nutrients (TDN) is 1 ~ 2kg, more preferably 1.4 ~ 1.6kg from one week before the start of hormone treatment for the target mammal. .

Induction of hypotrophy was performed as follows.

In the control group, 1 kg of mixed feed, 2.5 kg of high nutrition, 0 kg of low nutrition were fed. More detailed feeding conditions are shown in Table 1 below.

Table 1 shows the feed intake and TDN (plasticizing nutrient total amount) according to the nutrition level of the blank cow.

division Feed rate (kg) TDN (kg) Compound Feed Forage (straw straw) Compound Feed Forage (straw straw) Sum Control One 4 0.7 1.52 2.22 High nutrition 2.5 4 1.75 1.52 3.27 Low nutrition 0 4 0 1.52 1.52

Table 1 is based on the Korean specification ( www.nias.go.kr , 2007), based on the 450kg cow cow. The TDN of the compound feed used in this experiment was 1.75kg when ingested 2.5kg per day on a 70% basis, and the TDN of forage (straw straw) was 38% at 1.52kg TDN when ingested at 4kg (daily).

The total amount of plasticized nutrients (TDN) can be obtained from the following formula.

Total amount of plasticized nutrient (TDN) = amount of plasticized protein (DCP) + amount of plasticized fiber (DCFib) + amount of plasticized NFE (DNFE) + [amount of plasticized crude fat * 2.25]

Induction of hyperovulation using low nutrient status of empty cows

According to one embodiment, the low nutrition feeding period may be 20 to 25, more preferably 23 days.

The overovulation program of blank cows is shown in FIG. 1. Feeding for each test group took 23 days from 1 week before the start of hyperovulation (CIDR) to egg collection.

Induction of hyperovulation is as follows.

Hormonal treatment was performed by vaginal insertion of progesterone releasing intravaginal device (CIDR-plus, InterAg, New Zealand) and estradiol benzoate (EB) 1 mg (SY Esrone) , Samyang, Seoul, Korea) and 50 mg of progesterone (SY Ovaron, Samyang) were injected intramuscularly, and from day 4 28FS of follicle stimulating hormone (FSH, Antorin, 2AU = 1 ml, Kawasaki Mitaka, Japan) Loss splits (5, 5, 4, 4, 3, 3, 2, 2) were injected at 12 hour intervals over 6 days after progesterone release (CIDR) insertion and prostaglandins (PGF2, Lutalyse ^™ , Phamacia Co., Belgium was intramuscularly injected at 25 mg and 15 mg intramuscularly and the progesterone release device was removed. The artificial insemination was performed before and after 48 hours after prostaglandin (PGF2) injection, and 23 times of semens were performed at 12-hour intervals. After the first artificial insemination, 100 g gonadorelin (Gonadolerin, GnRH, Fertagyl, Intervet, Holland) Was injected into the muscle. Fertilized eggs were harvested 7 days after artificial insemination, and the perfusion solution for recovery was recovered by non-surgical method using a 3-way catheter using embryo collection medium (Embryo Collection Medium, Agtech, BiolifeTM, USA).

According to one embodiment, the egg is preferably carried out within 24 hours after low-nutrition feeding during the period, which is a low-nutrition state represented by the low-nutrition treatment of the present invention is temporary, the original form after 24 hours after egg collection Because it can be recovered.

Induction of hypotrophy Chaotic result

Experimental results of control, high and temporary low nutrition treatments for 23 days from 1 week before egg ovulation to the day of ovulation are shown below.

In case of fertilized eggs, the total number of fertilized eggs was 8.562.11 in control and 10.672.00 in high nutrition treatment, and 14.762.11 in low nutrition treatment. The results are shown in Table 2 below.

Treatment Compound Feed
Feed amount (kg) Disclosure Recovered
(Average / two) Number of implantable fertilized eggs (average / two) Implantable fertilized egg
ratio(%) Control One 16 8.56 ± 2.11 4.63 ± 0.98 ^a 54.08 High nutrition 2.5 9 10.67 ± 2.00 7.33 ± 2.18 68.69 Low nutrition 0 17 14.76 ± 2.11 10.94 ± 1.91 ^b 74.11 Average 11.52 ± 1.31 7.76 ± 1.05 67.36

In the experiment, the atrophy ovary (14 heads) was found to have a distribution of 3 controls, 6 high-nutrition treatments and 5 low-nutrition treatments.

The different alphabets in Table 2 indicate that there is a significant difference in the statistics between implantable fertilized eggs (p <0.05). As shown in Table 2, it can be seen that the number and ratio of implantable fertilized eggs in the low nutrient treatment is relatively high.

In addition, as shown in Table 2, the number of transplantable eggs was 4.63 control, 7.33 high nutrition, 10.94 low nutrition, and the number of low nutrition was higher, 54.08%, 68.69% and 74.11% of the temporary low-nutrition group had a significant number of implantable fertilized eggs and a high recovery rate.

Comparison of the hyperovulation reaction rate according to the above low nutrition treatment is shown in Table 3 below. In the control and low-nutrition diets, the rate of hyperovulation was 84.21 and 77.27%, which was low in atrophic ovaries (3/19 and 5/22), but in the high-nutrition (6/15), the rate of over-ovulation was 60%. The high nutrient treatment showed lower overovulation effect (overovulation reaction rate) than the low nutrition treatment.

Treatment Compound Feed
Feed amount (kg) Public announcement
(two) Poached water
(two) Atrophy
(two) Hyperovulation reaction rate
(%) Control One 19 16 3 84.21 High nutrition 2.5 15 9 6 60.00 Low nutrition 0 22 17 5 77.27 system 56 42 14 75.00

Table 4 below shows the results of analyzing the number of implantable fertilized eggs according to the number of corpus luteum and the ratio thereof. It can be seen that when the number of the corpus luteum is more than 10, the proportion of implantable fertilized eggs is high.

Corpus luteum Disclosure Recovered
(Mean standard error) Implantable fertilized eggs
(Mean standard error) Implantable fertilized egg
ratio(%) <10 11 5.36 ± 1.30 2.73 ± 1.23 ^a 50.93 ≥ 10 34 14.29 ± 1.45 9.50 ± 1.28 ^b 66.48 Atrophy 14 0 0 0 system 59

(Atrophy ovary (14 heads): 3 control, 6 high and 5 low)

Different alphabets indicate significant differences in statistics between implantable fertilized eggs (p <0.01)

Table 5 below shows the results of analyzing the number and ratio of implantable fertilized eggs according to the number of corpus luteum by the nutrition level of the blank cow.

Corpus luteum division Disclosure Recovered
(Mean standard error) Implantable fertilized eggs
(Mean standard error) Implantable fertilized egg
ratio(%)
10
Control 5 4.80 ± 1.88 3.20 ± 1.59 66.66 High nutrition 3 8.00 ± 2.65 4.00 ± 4.00 50.00 Low nutrition 3 3.67 ± 2.67 0.67 ± 0.33 18.18
10
Control 10 11.20 ± 2.98 5.70 ± 1.27 50.89 High nutrition 6 12.00 ± 2.67 9.00 ± 2.56 75.00 Low nutrition 17 16.00 ±  1.83 10.88 ±  1.93 68.01
Atrophy
Control 3 0 0 0 High nutrition 6 0 0 0 Low nutrition 5 0 0 0 system 58

Referring to Table 5, the proportion of implantable embryos in the control group was high at 66.66% in the lower luteal count but lower in 18.18% in the low nutrition group. This result was due to the lack of the number of subjects and there was no significant difference.

However, in 10 or more corpus luteum, the percentage of implantable fertilized eggs was 75% and 68% in the high and low nutrient treatments compared to 50.89% in the control, and the number of implantable fertilized eggs was 9 and 10.88, respectively. In atrophy ovaries, the recovery was 0% regardless of treatment.

Considering the above results, it can be seen that the number of eggs and implantable fertilized eggs was higher than that of the control and the high nutrition during the transient ovulation treatment by the temporary low nutrition specification. Therefore, the present invention is judged to be an efficient chaos technology with high overovulation reaction efficiency with less atrophy ovaries. According to the present invention, it is possible to increase the number of implantable fertilized eggs for Hanwoo blank cows. Through this, it is possible to reduce the effort, processing cost, and feed cost according to the treatment of empty ovulation and recovery of fertilized eggs, and it is possible to improve high quality cow breeds with high quality grade.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (5)

A method for producing bovine body fertilized egg, characterized in that by inducing over-ovulation in the temporary low nutritional status of the cow.
The method of claim 1, wherein the low-nutrition state is induced by low-nutrition feeding so that the total plasticized nutrient total amount (TDN) of the cow is 1 ~ 2kg.
The method of claim 2, wherein the low-nutrition feeding period is 20 to 25 days.
The method of claim 2, wherein the egg is produced within 24 hours after the low nutrition feeding.
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