JPH06113697A - Selection of bovine egg enabling embryogenesis - Google Patents

Abstract

PURPOSE:To select a cumulus-ovum complex (COC) having embryogenetic capability from COC obtained from the ovary of a cow after artificial fertilization. CONSTITUTION:A cumulus-ovum complex (COC) collected from the ovary of a cow is inspected with a microscope to classify the complexes into the following four types and the COC belonging to type 2 or type 3 are collected. Type 1: COC having uniform ooplasm covered with a relatively firmly bonded multi- layer granulosa cell structure. Type 2: COC having ooplasm covered with a firmly bonded multi-layer granulosa cell structure and containing a number of relatively large-sized oil spheres uniformly distributed in the ooplasm. Type 3: COC having ooplasm covered with a dark multi-layer granulosa cell structure appeared to have relatively weak bonding force and containing a number of relatively large oil spheres distributed in the ooplasm forming irregular clusters. Type 4: The bond of the granulosa cells covering the ovum is almost lost, the granulosa cells are nearly in a diffused state and the individual granulosa cells are radially distributed in a jelly substrate.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a Cumulus Oocyte Complex (Cumulus Oocyte Complex) capable of embryogenesis by artificial fertilization.
Hereinafter, it may be abbreviated as COC).

[0002]

2. Description of the Related Art A large number of follicular ova are present in the ovaries of cattle that have been killed at a slaughterhouse, and if they can be collected and artificially fertilized and then grown in the bovine mother, the cattle will be extremely efficient. Can be bred. However, conventionally, even if the ovary of a bovine slaughtered in a slaughterhouse has a large number of follicular ova in its tissue, a basic technology for maturing, fertilizing, and developing the ovarian ova in the maturing process is established. Since it has not been done, it was almost impossible to use them effectively.

However, only recently, the cumulus (or granulosa) cell Kajihar is the first step to establish the basic technology.
a et al. (1987) (Reference 1), Goto et al. (1988) (Reference 2), Fukuda et al. (1990) (Reference 3), Ellington et a.
l., (1990) (References 4 and 5) or oviductal epithelial cells Eyest
one et al., (1987) (Reference 6), Eystone and First (198
9) (Reference 6) and follicular egg [or cumulus-egg complex (CO
It was reported that it is possible to efficiently produce a blastocyst that can be transplanted by co-culturing C)] and then in vitro fertilization. De Loos et al., (1989) (Reference 7) reported that morphological typing of COC and developmental capacity are closely related. However, their morphological classification is highly subjective and lacks concrete support, so what type of COC is still the most effective CO
It can be said that the objective criteria for whether or not it is C have not been established.

[0004]

Therefore, the present invention provides a method for easily and objectively selecting from among the COCs collected at a slaughterhouse, the COCs highly likely to lead to embryogenesis by artificial fertilization. It is what

[0005]

In order to solve the above problems, the present inventor has conducted various studies on the relationship between various properties of eggs in COC and embryogenicity after artificial fertilization. It can be divided into 4 types by observation, the combination of these types and the size of the naked egg obtained from COC, and the observation results of illuminance, transparency and polarization in the microscopic observation of the protoplasm and after artificial fertilization. The present invention has been completed based on the completely new finding that it has a high correlation with the embryogenic ability of the.

Accordingly, the present invention provides C extracted from adult ovary.
The following four types of OCs were observed under a microscope: Type 1; composed of egg cells with a uniform egg cytoplasm surrounded by a relatively tightly bound multi-layered granulosa cell structure, and its overall COC optical The color is bright and transparent; type 2; composed of egg cells in which a large number of relatively large oil spheres are uniformly distributed in the egg cytoplasm surrounded by a tightly bound multi-layered granulosa vesicle structure And its overall COC optical tone is slightly darker and less transparent than Type 1; Type 3; Compared to its egg cytoplasm surrounded by a dark multi-layered granulosa cell structure that appears somewhat weakly bound. Large oil globules are composed of egg cells distributed irregularly in clusters, and the overall COC optical color tone is darker and less transparent than type 2; and type 4; egg cells Most of the granulosa cells surrounding the cells lose their connectivity and spread, and the granulosa cells are distributed radially in the jelly-like matrix. The central part of the oocyte is dark and the peripheral part is composed of transparent oocytes. , And its overall COC optical tones are dark and opaque;
Alternatively, it relates to a method of selecting a COC belonging to type 3.

[0007]

[Detailed Description] The present invention collects ova from an ovary of a bovine slaughtered by artificially fertilizing the ovum and growing the ovum in a cow to breed the bovine. Highly embryogenic COC by observing COC under a microscope
It is intended to improve the success rate of breeding, and the selection criterion is to select a COC belonging to the type 2 or type 3 among the 4 types by observing the COC with a microscope.

Specific micrographs of these types 1 to 4 are shown in FIGS. 1 to 4, respectively. The feature of the present invention is that the above four types are statistically well correlated with other observation results, that is, the size, brightness, transparency and polarizability of the naked egg, and the morphogenesis process of the fertilized egg after fertilization and It has a high correlation with the embryogenicity, and is highly accurate and highly objective as a criterion.

Below, it will be explained based on the experimental results that the above-mentioned criteria are well correlated with other parameters and embryogenicity. Experiment 1 . Collection and typing of COCs Adult cattle ovaries collected within 1 hour after slaughter were put in a plastic bag, transported around to the laboratory while being kept warm with warm water of 32 ° C-35 ° C, and used for the experiment. Ovary TCM199
25mM HEPES, polyvinyl alcohol (100μ
g / ml), 1.25 mM sodium pyruvate, heparin (15 μg / ml) and the antibiotic gentamicin (10
It was transferred into a 100 mm diameter petri dish containing a medium containing μg / ml). The ovaries halved with surgical scissors were finely incised with a multi-surgery surgical knife, and COCs were collected and used in the experiment.

COCs have been classified into four types based on the following criteria. Type 1; this type of COC was composed of egg cells with a uniform egg cytoplasm surrounded by a relatively tightly bound multi-layered granulosa cell structure as shown in FIG. Moreover, the optical color tone of the overall COC was bright and transparent.

Type 2; This type of COC is 2 in FIG.
As shown in Fig. 3, the egg cell was surrounded by tightly bound multi-layered granulosa vesicle structures, and consisted of egg cells in which a large number of relatively large oil spheres were uniformly distributed. Also, the optical color tone of the overall COC was slightly darker and less transparent than the type 1. Type 3; This type of COC has a large number of relatively large oil globules in its egg cytoplasm surrounded by a dark multi-layered granulosa cell structure whose binding is somewhat weak, as shown in 3 of FIG. It consisted of egg cells distributed in a regular mass. The optical color tone of the overall COC was darker and less transparent than the type 2.

Type 4; This type of COC is 4 in FIG.
As shown in, the granulosa cells surrounding the egg cells are almost lost of their connectivity and diffuse, and the granulosa cells are radially distributed in the jelly-like matrix, and the central part of the oocyte is dark and its periphery is dark. Was composed of transparent egg cells. And the optical color tone of the overall COC was dark and opaque.

Experiment 2 Observation of naked ova 1) Method for preparing naked ova The naked ova were prepared with COC of 0.
After immersing in TCM199 containing 2% hyaluronidase and confirming that the cumulus cells are scattered to some extent, prepare a pipette with a diameter slightly larger than the diameter of the egg, connect the mouthpiece to it and operate the mouthpiece. By doing so, the COC suction was repeatedly adjusted.

2) Method for preparing and recording observation sample of naked egg a ) Preparation of live egg observation sample Vaseline, lanolin and paraffin were mixed at a ratio of 2: 2: 1 (weight ratio) (Warlap), and Is melted by heat, and a spot of 4 points is created on the slide glass so that a cover glass of 22 mm × 22 mm is placed on the mixed melt.
Place the sample to be observed in the center together with the culture solution, cover the cover glass with care so that the sample is located in the center as much as possible, press the cover glass lightly so that the sample does not collapse, and the culture solution does not dry around As described above, the sample was sealed with a transparent nail polish enamel and used as an observation sample.

B) Preparation of Fixed Oocyte Observation Specimen As in the case of a living egg observation specimen, a slide glass having four Warlap spots was prepared, and the specimen was placed at the center of the four spots, and the Carnoy's fixative was prepared. Fix overnight with, stain with acetoorcein for 5 minutes, then wash with water, cover with a cover glass while being careful to keep the sample in the center as much as possible, gently press the cover glass so that the sample does not collapse, and keep the surroundings transparent. It was sealed with a nail polish enamel and used as a specimen for observation.

C) Observation record A transmission type Marski type differential interference microscope (Olympus Model BH2-USD) was used for observing the sample. In addition, automatic exposure type photographic equipment (O
The Lympus Model C-35AD-4) was used. 3) Statistical processing A χ ² test was performed to confirm the significance in each experiment.

The results are shown in FIG. 2 and Table 1.

[Table 1]

As shown in 1 of FIG. 2, the characteristics of the naked egg obtained from the type 1 COC are that the diameter of the egg is slightly smaller than that of the other three types and the structure of the ooplasm is uniform. , The oil tone was hardly seen and the color tone was bright. Therefore, it can be said that it is bright and transparent as a whole.

As shown in 2 of FIG. 2, the characteristics of the naked egg obtained from the type 2 COC are that the diameter thereof is slightly larger than that of the type 1, and the structure of the oocyte is uniform and the oil droplets are Was evenly distributed over the whole egg, and the color tone was darker than that of type 1. Therefore, it can be said that the whole egg is slightly dark and opaque. The characteristics of the naked egg obtained from type 3 COC have the same diameter as type 2 as shown in 3 of FIG. Had a relatively large agglomerate and were evenly distributed. Different from type 2, eggs or the zona pellucida showed a very dark color tone. Therefore, it can be said that the whole egg is dark and opaque.

As shown in 4 of FIG. 2, the characteristics of the naked egg obtained from the type 4 COC are that the diameter of the egg is the same as that of types 2 and 3, but the oocyte is a structure containing oil droplets. Since the object is localized in the center of the egg, the center is opaque and dark,
Intracellular structures were not found around the oocyte. Therefore, it can be said that it has the same brightness as the type 1 as a whole but is opaque.

When the same eggs as these eggs were observed by changing the background differential interference colors, as shown in 1, 3, 2, 3, 3, 3 and 4 of FIG. A polarized color is observed in the plasma membrane of the egg (1 in FIG. 3) and the plasma membrane of the type 4 egg (4 in FIG. 3), but the type 2 egg (2 in FIG. 3) and the type 3 egg (see FIG. 3) It is clear that no polarized color is observed in the plasma membrane of 3) of 3). Such optical facts mean that the physical properties of the plasma membrane differ depending on the type of egg.

Experiment 3 Oocyte maturation process Next, the COCs were matured and cultured, artificially fertilized, and fertilized eggs were cultured to observe the possibility of embryogenesis, and the correlation between the COC typing and the embryogenic potential was examined. 1) COC maturation culture method The collected COCs were washed twice with TCM199 solution, and about 30 COCs each were 350 μl of maturation medium TCM19.
Liquid 9 was added to sodium bicarbonate (2 mg / ml), 10% fetal bovine serum, and each drop of maturation medium was covered with mineral oil to prevent drying. 3 for mature culture
It was carried out for 21 to 23 hours in a humidified incubator of 5% carbon dioxide / 95% air at 8.5 ° C.

2) In vitro fertilization method Commercially available frozen Japanese black cattle semen (0.5 ml) is thawed in warm water at 35 ° C., the semen is placed in a centrifuge tube, and cow containing 5 mM caffeine and heparin (15 μg / ml) is added. BO medium without serum albumin (BSA) (6 ml) (Brackett and O
Liphant; Biol. Reprod. 12 : 260-274, (1975) (Reference 8) was added and mixed. 2,200 for 7 minutes with this mixture
Centrifuge by rotation and discard the supernatant. This sperm washing operation was performed once more in the same manner. Then, the concentration of sperm was measured using a hemocytometer and adjusted to 10 ⁷ cells / ml. 50 μl of this sperm fluid was added to 50 μl of BO medium containing 5 mM caffeine and fatty acid-free BSA (10 mg / ml). Eggs that have been matured and cultured for 21 to 23 hours are washed three times with the above-mentioned medium, then put into a sperm suspension, and the eggs are incubated at 38.5 ° C for 5 hours.
6% in humidified incubator of% carbon dioxide / 95% air
Fertilization was carried out by culturing for a period of time. The concentration in the BO solution at the time of fertilization was 5 × 10 ⁶ sperm / ml, 5 mM caffeine,
They were heparin (7.5 μg / ml) and BSA (5 mg / ml).

3) In vitro fertilization culture method Fertilized egg, TCM199 solution, insulin (5 μg / ml)
After washing twice with the development medium containing aprotinin (500 ng / ml), 350 μl of each spot of the development medium was prepared, and the fertilized egg was transferred into this development medium. The petri dish contains TCM at a final concentration of 150 μg / ml of type 1 collagen to promote the growth of cumulus / granulosa cells.
199 solution was prepared, incubated at room temperature for 1 hour, and then used for culture. Fertilized eggs are 24 in each development medium
After time culturing, cumulus / granulosa cells adhering around the fertilized egg were naked using a capillary-like pipette. The resulting fertilized naked eggs were transferred to 350 μl of each test-like development medium and cultured at 38.5 ° C. in a humidified incubator with 5% carbon dioxide / 95% air.

The observation results were as follows. 1) Judgment of each meiotic stage of the egg The observed meiotic stage of the nucleus of the egg is the state of each chromosome as a standard, and the nucleoli phase, the chromosome condensation phase, the migration phase, and the first meiosis. It was classified into eight stages: metaphase, late phase, terminal second meiotic prophase, and metaphase. In addition, those that could not be classified into any stage were judged as undecidable.

A) Before Maturation Culture The meiotic stages of the nucleus of each type of oocyte before maturation culture are shown in Table 2. The results in Table 2 clearly indicate that: In other words, about type 1 ovum, 61/62 is mostly in the follicular phase and type 2
For type 3 and 3 ovum, 7/50 and 2/38 were in the follicular phase, and 43/50 and 3 were in the more advanced stages of chromosome condensation and migration.
Many were 4/38. For type 4 eggs,
No germinal vesicles were observed at all, 1/33 in chromosome condensation / migration and 6 in the first metaphase.
/ 33 is recognized to some extent. Furthermore, with Type 4, it was difficult to judge the stage of the nucleus, and there were many cases where it was impossible to judge 26/33.

[0027]

[Table 2]

B) Post-Maturation Culture The stages of meiosis of the nuclei of each type of egg after mature culture are shown in Table 3. The results in Table 3 clearly indicate that: That is, 1 type 1 egg has reached the second stage of meiosis, which is a state in which maturation is completed.
There is almost no / 51, and 25/51 of about half of all the eggs used in the experiment are in the metaphase of the first meiosis,
In addition, 18/51 more ova were found in which the disintegration of the follicular vesicles did not occur compared to other types of ova. In addition, it can be confirmed that 60% or more of type 2, 3 and 4 eggs have reached the second metaphase of meiosis (the values are 37/56 for type 2 eggs and 67 / for type 3 eggs, respectively). 75, and 27/39 for type 4 eggs).

As a result, when comparing these pre-maturation culture data and post-maturation culture data, the type 1 egg is in a state called a mature egg even when it is matured (that is, at least the second meiotic metaphase). It is an egg of a degree of maturity that does not reach the advanced stage), and the type 2, 3 and 4 eggs are called mature eggs when they are matured and cultured (that is, at least advanced to the second meiotic metaphase). ) Can be said to be an egg of maturity.

[0030]

[Table 3]

2) Differences in developmental ability of typed COC-derived ova The results of the effects of egg types on development are shown in Table 4. From Table 4, the incidences after the 2-cell stage are 11% and 71% for types 1, 2, 3 and 4, respectively.
85% and 74%, types 2, 3 and 4 showed a statistically significant value (P <0.001) higher than type 1, and the same tendency was observed in the 8-cell stage and the morula stage. You can see that However, only the type 2 and 3 eggs that reached the blastocyst were 11% and 9 respectively.
%Met. And it is clear that the value shows a statistically significant (P <0.05) high value with respect to types 1 and 4.

[0032]

[Table 4]

Discussion As shown by 1 in FIG. 1, the egg of type 1 is slightly small in size and has a bright color tone. It is said that the color tone of the egg is related to the lipid and glycogen content, and the lipid and glycogen are taken in with the development of the egg, and the color tone becomes peculiar to each species. In the case of bovine ova, it is rich in lipid, contains little glycogen, and is close to black. Therefore, a bright color means that lipid uptake has not been carried out so much. Further, the nuclear disintegration ability, which is one of the maturation abilities, is strongly related to the degree of oocyte development, and it is considered that the ability is expressed when the oocyte development is completed.

In this experiment, type 1 ova had the lowest rate of collapse of the follicular follicles after maturation culture, as compared to other types of ova (Table 4). Considering these facts, it is considered that the type 1 ovum is an immature ovum in the early to middle stages of the oogenesis process in the ovary. Therefore, even if in vitro fertilization is carried out after maturation culture, development does not proceed because maturation is not completed. However, after maturation culture, about half of the eggs were in the first metaphase of meiosis, but since these eggs stopped at this stage, it is unknown whether they are in the process of meiosis. Therefore, it is necessary to extend the maturation culture time and investigate whether or not the second metaphase of meiosis is reached.

Eggs of types 2 and 3 were premature (Table 2),
After maturation (Table 3), the nuclei stage and the occurrence rate show almost the same tendency (Table 4), and can be said to be an egg that can be effectively used in the culture system that is generally performed at present such as this experiment. The reason why many of the nuclei before maturation were those in the chromosomal enrichment stage, which was advanced from the follicular vesicle stage, was that although the experiment was carried out while cooling with ice, it took 3 to 4 times until COC was taken out from the cells and fixed Probably because it takes time. In addition, it is said that the resumption of meiosis of an egg occurs not only after it is taken out from the follicle but also due to the death of the individual. It took about 6 hours.

It has been reported that in bovine ova, in vivo and in vitro, follicular blast collapse occurs 4 to 6 hours after meiosis resumes (Hyttel et al., 198).
6 (Reference 9); Kruip et al., 1983 (Reference 10)). Therefore, it may be natural that the stage of meiosis of the nucleus before the maturation of the egg in this experiment is the chromosomal enrichment phase or migration phase. But,
In the type 1 ovum, the stage of meiosis of the same premature nucleus was almost in the follicular phase. This represents a difference in responsiveness to the same stimulus for resumption of maturation,
It can be said that type 1 eggs are less reactive than type 2 and 3 eggs.

The stage of meiosis of the nucleus before maturation of type 4 eggs was 6/33 in the first meiotic metaphase, which was further advanced than the migration phase (Table 2). To reach the first metaphase of meiosis, about 1
2 to 14 hours are required. Therefore, even if meiosis starts immediately after slaughter, it is not possible to reach the first metaphase of meiosis until it is fixed.
It is considered that the ova of the ovum had been stimulated with hormones and the like without ovaries, and that the ova had already been activated and meiosis had started before being slaughtered. In fact, in another experiment, it was also observed that the first polar body was released in the type 4 eggs immediately after the collection.

[0038] Furthermore, many type 4 eggs contained many of them whose meiotic stage of the nucleus could not be determined (26/33) (Table 2). And some of these eggs were apparently not normal chromosome images. It is unlikely that these eggs were also denatured after they were removed from the ovaries, and it is speculated that these eggs are destined to regress in the ovaries. Considering these facts, it can be inferred that the type 4 egg is an egg just before ovulation that started meiosis in the ovary or an egg that regresses without ovulation.

Looking at the occurrence of type 4 eggs,
Up to morula, the incidence was almost the same as that of type 2 and 3 eggs, but none reached the blastocyst stage (Table 4). It seems that the type 4 development is not the regressive egg but the egg just before ovulation.
If this assumption is correct, it means that it has reached the second metaphase of meiosis in a shorter time than other types of eggs because it has advanced to a stage earlier than other types before the start of maturation culture. Inferred. Therefore, it is considered that the ovum is aging because the maturation culture time of 22 hours is too long, and this aging may be one of the reasons why it cannot reach the blastocyst.

According to the report of Kajihara et al., (1987) (Reference 1), an egg that has been cultured for a long period of time has the same embryogenesis rate as that of an egg with a short maturation culture time up to the morula but The rate of cell formation tends to be low. This seems to imply that type 4 eggs may be able to develop into blastocysts if the maturation time is shortened.

When the interference color lever was gradually moved with a Nomarski differential interference microscope and the cytoplasm was observed in detail, the polarization color due to the fluctuation of the birefringence of the plasma membrane was observed in the periphery of the cytoplasm of type 1 and type 4. However, the polarized color cannot be observed in the cytoplasm of type 2 and type 3 (Fig. 3). This is because the type 1 egg and type 4 egg membranes are
It has been shown that the physical properties of the membranes of the egg of Type 1 and the egg of Type 3 are different. In general, biochemical data on biomembranes show that protein uptake into lipid bilayers, desaturation of fatty acids or length of bicarbonate chains alters the birefringence of the membrane,
Since it is known to affect the polarization property, the difference in the polarization property in the membrane found in this experiment is due to some change in the physical property of the plasma membrane during the process of oogenesis, maturation, development and regression. Can be considered to indicate that there is a possibility.

From the above experimental data, it is clear that even if it is an optical microscope, the morphological characteristics and the polarization of the membrane that have already been shown are used as indicators to identify eggs with different rates of maturity and development. It has been shown that it is possible to sort. Table 5 shows a summary of excellent egg selection criteria for such effective utilization of eggs in the ovary.

[0043]

[Table 5]

Reference (1) Y. Kajihara, K. Goto, S. Kosaka, Y. Nakanish
i and K. Ogawa (1987) In vitro fertilization of bov
ine follicular oocytes and their development up to
hatched blastocysts in vitro.Jpn.J.Anim. Reprod.
33 (4), 173-180. (2) K.Goto, Y.kajihara, S.Kosaka, M.koba, Y.Naka
nishi and K. Ogawa (1988) Pregnaucies after co-cult
ure of cumulus cells with bovine embryos derived f
rom in-vitro fertilization of in-vitro matured fol
Licular oocytes. J. Reprod. Fertil., 83 ; 753-758. (3) Y. Fukuda, M. Ichikawa, K. Naito and Y. Toyo
da (1990) Birth of normal calves resulting from bo
vine oocytes matured, fertililized, and cultured w
ith cumulus cells in vitro up to the blastocyst st
age. Biol. Reprod. 42, 114-119.

(4) JEElligton, EWCarney, PBFa
rrell, MESimkin and RHFoote (1990) Bovine 1-2-
cell embryo development using a simple medium in t
hree oviduct epithelial cell coculture systems. Bi
ol. Reproce., 43 ; 97-104. (5) JEEllington, PBFarrell, MESimkin, RH
Foote, EEGoldman and A.B.MeGrath (1990b) Developm
ent and Survival after transfer of cow embryos cul
fured from 1-2-cells to moralae and blastocysts in
rabbit oviducts or in a simple medium with bovine
oviduct epithelial cells. J.Reprod.Fert., 89 ; 2
93-299. (6) WH Eyestone and NL First (1989) Co-cul
ture of early cattle embryos to the blastocyst sta
ge with oviducal tissue or in conditioned medium.
J. Reprod. Fert. 85, 715-720.

(7) F. De Loos, C. Van Vliet, P. Van
Maurik and TAM Kruip (1989) Morphology of imm
ature bovine oocytes. Gamete Res. 24, 197-204. (8) BG Brackett and G. Oliphant (1975) Capaci
tation of rabbit spermatozoa in vitro. Biol. Repro
d. 12, 260-274. (9) P. Hyttel, KP Xu, S. Smith and T. Greve
(1986) Ultrastructure of in-vitro oocyte maturatio
n in cattle. J. Reprod. Fert. 78, 615-625. (10) TAM Kruip, DG Cran, TH Beneden an
d SJ Dieleman (1983) Structual changes in bovin
e oocytes during final maturation in vivo.Gamete
Res. 8, 29-43.

[Brief description of drawings]

FIG. 1 is a photomicrograph showing COC types 1 to 4, which is a photograph instead of a drawing showing the morphology of living things.

FIG. 2 is a type 1 of a naked egg obtained from COC.
4A to 4D are micrographs of drawings, which are photographs instead of drawings showing the morphology of living things.

FIG. 3 is a naked egg type 1 obtained from COC.
4A to 4C are polarization micrographs of FIGS. 4A to 4C, which are photographs instead of drawings showing the morphology of living things.

Claims (1)

[Claims] 1. A cumulus-egg complex (COC) collected from an adult ovary is observed under a microscope and has the following four types: type 1; homogeneous surrounded by relatively tightly bound multi-layered granulosa cell structures. It is composed of egg cells with various egg cytoplasms, and the optical color tone of the overall COC is bright and transparent; type 2; in the egg cytoplasm surrounded by tightly bound multilayer granulosa vesicle structures, It is composed of many uniformly distributed egg cells with relatively large oil globules, and the optical color tone of the overall COC is slightly darker and less transparent than type 1; type 3; its binding is somewhat weak The oocyte, surrounded by dark visible multi-layered granulosa cell structures, is composed of egg cells in which a large number of relatively large oil globules are irregularly formed in clusters and distributed. The color tone is Thailand It is much darker and less transparent than type 2; and type 4; the granulosa cells surrounding the egg cells lose most of their connectivity and diffuse, and the granulosa cells are radially distributed in the jelly-like matrix. The central part of the cytoplasm is dark and the peripheral part is composed of transparent egg cells, and the optical color tone of the overall COC is dark or opaque;
Alternatively, a cumulus-egg complex belonging to type 3 is selected.