Implantable implantable procreation device and method for preparing embryo transfer

Abstract

We propose an internally implantable production device that can culture fertilized eggs. By inserting this device into the body of a cow, the use of expensive carbon dioxide culture equipment becomes unnecessary, and the rate at which fertilized eggs reach the blastocyst stage is increased, making the process more efficient. [Solution] The device comprises an outer shell consisting of a substantially cylindrical outer shell casing 21 having a recessed end and a casing lid 23 provided on the open end side of the outer shell casing 21 to create a closed space over the recess; a culture container 10 housed inside a storage cavity formed by the closed space of the outer shell; and a holder 24a positioned inside the storage cavity, which serves as a gap setting means to set a gap around the culture container 10 by defining the position and orientation of the culture container 10 inside the storage cavity. Fertilized eggs are stored inside the culture container 10 together with a culture fluid 8, and the outer shell containing the culture container 10 is placed inside the body of a cow via the cow's vagina, and the fertilized eggs are cultured using the cow's body temperature.

Description

[Technical Field]

[0001] The present invention relates to an internal implantable production device for inserting and culturing fertilized or unfertilized eggs of a cattle (bovine) that have been fertilized in vitro, and to a method for preparing a cattle fertilized egg for transplantation using this internal implantable production device. [Background technology]

[0002] Some of the present inventors have already proposed a technique for culturing in vitro fertilized bovine eggs without using expensive carbon dioxide culture devices (incubators) (see Patent Document 1). Furthermore, the present inventors have proposed a calving detection system in which a circuit housing capsule containing a transmitter that emits radio waves is inserted into the vagina of an animal, and radio waves are emitted from the transmitter inside the vagina and received by a receiving device located externally (see Patent Document 2).

[0003] Patent Document 1 proposed a method for producing in vitro fertilized eggs (IVF) that can be transplanted into recipient cows (transplant cows) by sequentially going through the maturation culture stage, fertilization (insemination) stage, and developmental culture stage using transvaginal oocytes (OPU) derived from oocytes, without using a carbon dioxide culture apparatus, and performing the entire process in a predetermined space. However, the invention disclosed in Patent Document 1 proposed a configuration in which the culture test tube directly comes into contact with bodily fluids in the vagina, which presented a hygienic problem. Therefore, the present inventors began investigating a structure in which the culture test tube does not directly come into contact with the vagina. However, new problems arose, such as what material should be chosen for the vaginal insertion housing that contains the culture test tube to be highly compatible with the cow's vagina, or that variations in the operator's skill and method regarding the positional relationship between the vaginal insertion housing and the culture test tube would result in poor reproducibility of the blastocyst formation rate. To widely disseminate a simple in vitro fertilization embryo production technology that does not require a carbon dioxide culture system to producers (farmers), a standardized technology with improved reproducibility and reliability, independent of the farmer's skills, is necessary.

[0004] The birth detection system proposed in Patent Document 2 has a structure in which a circuit housing capsule is housed in an outer shell and the outer shell is inserted into the vagina of an animal. The inventors focused on a structure in which the outer shell proposed in Patent Document 2 can also be adapted for use as an internally insertable production device for culturing fertilized or unfertilized eggs. That is, they realized that if the main components proposed in Patent Document 2 could be developed into an internally insertable production device, it would be possible to simplify and standardize the in vitro fertilized egg production method, which is one of the "3S principles of rationalizing production." Furthermore, they realized that by simplifying and standardizing, which are the basic principles of the 3S principles, it would be possible to improve the reproducibility and reliability of the in vitro fertilized egg production method using an internally insertable production device, and to promote the widespread use of a simple in vitro fertilized egg production technology that does not use a carbon dioxide culture device. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2023-100120 [Patent Document 2] Japanese Patent Publication No. 2023-145410 [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention proposes a hygienic, internally insertable production device suitable as a new technology to be widely disseminated to producers (farmers), which can reproducibly culture fertilized or unfertilized eggs of in vitro fertilized cattle. By inserting this internally insertable production device into the body of a cattle, the use of expensive carbon dioxide culture equipment is eliminated, and the invention aims to provide a method for preparing embryos for transfer that can stably increase the rate at which fertilized eggs reach the blastocyst stage, allowing for efficient transfer to the embryo transfer process to recipient cows (transfer cows). [Means for solving the problem]

[0007] To solve the above problems, the gist of the present invention is an internally insertable production device comprising: (a) an outer shell portion comprising a substantially cylindrical outer shell housing having a recessed end and a housing lid provided on the open end side of the outer shell housing to create a closed space over the recess; (b) a culture container housed inside a storage cavity formed by the closed space of the outer shell portion; and (c) a gap setting means disposed inside the storage cavity, which sets a gap around the culture container by defining the position and orientation of the culture container inside the storage cavity. The internally insertable production device according to the first embodiment houses fertilized eggs or unfertilized eggs together with a culture fluid inside the culture container, and the outer shell portion housing the culture container is placed inside the body of a cow via the cow vagina, and the fertilized eggs or unfertilized eggs are cultured using the cow's body temperature.

[0008] A second aspect of the present invention is a method for preparing an embryo for transplantation, comprising the steps of: (a) preparing a culture vessel for use in the developmental culture process by placing a fertilized egg in a culture test tube together with a culture medium and covering the culture test tube with a test tube cap; (b) filling the culture vessel with a culture fluid by replacing the gas inside the culture vessel for use in the developmental culture process using a gas phase injector so that the composition of the culture gas phase is similar to that of the bovine body; (c) placing the culture vessel inside the storage cavity of the outer skin via a gap setting means so that it is in a predetermined orientation and position, setting a gap around the culture vessel using the gap setting means, and embedding a heat buffer sheath made of gas in this gap to constitute an internally insertable production device for the developmental culture process; and (d) placing the internally insertable production device inside the body of a bovine via the bovine vagina and performing the developmental culture process of the fertilized egg using the bovine body temperature. [Effects of the Invention]

[0009] According to the present invention, it is possible to propose a hygienic, internally insertable production device that is suitable for standardization of technology and can be widely disseminated to farmers, and that can culture in vitro fertilized bovine fertilized or unfertilized eggs with high reproducibility. Furthermore, by inserting this internally insertable production device into the body of a cow, it becomes unnecessary to use an expensive carbon dioxide culture device, and it is possible to provide a method for preparing embryos for transfer that can stably increase the rate at which fertilized eggs reach the blastocyst stage and efficiently proceed to the embryo transfer process to the recipient cow (transfer cow). [Brief explanation of the drawing]

[0010] [Figure 1A] This is a schematic top view illustrating the main components of an internally implantable production device according to the first embodiment of the present invention. [Figure 1B] This is a schematic cross-sectional view illustrating the main parts of the internally implantable production device according to the first embodiment. [Figure 1C] Figure 1C(a) is a schematic top view illustrating the general structure of the outer casing side of the implantable production device according to the first embodiment, and Figure 1C(b) is a left side view of the outer casing side structure corresponding to Figure 1C(a). [Figure 1D] Figure 1D(a) is a bird's-eye view of the housing lid that constitutes the outer shell of the implantable production device according to the first embodiment, Figure 1D(b) is a top view of the housing lid shown in Figure 1D(a), and Figure 1D(c) is a left side view of the housing lid corresponding to Figure 1D(b). [Figure 1E] Figure 1E(a) is a schematic cross-sectional view illustrating the assembled state in which a culture container is inserted into the through-hole of the gap setting means (holding device) that sets the gap Vgap of the internally insertable production device according to the first embodiment; Figure 1E(b) is a bird's-eye view illustrating the holding device shown in Figure 1E(a); and Figure 1E(c) is a bird's-eye view illustrating the culture container inserted into the through-hole of the holding device shown in Figure 1E(b). [Figure 1F] This is a schematic cross-sectional view illustrating the main parts of an internally implantable production device according to a first modified example of the first embodiment. [Figure 2] This is a schematic diagram illustrating a method for preparing bovine embryos for transplantation using an internally implantable production device according to the first embodiment. [Figure 3]This is an equivalent circuit illustrating the heat flow in an implantable production device according to the first embodiment. [Figure 4A] This figure shows the time course of the bovine body temperature over one day, corresponding to the maturation culture step, one of the three steps in the in vitro fertilization of bovine eggs using the internally inserted production device according to the first embodiment. [Figure 4B] This figure shows the change in temperature at the bottom of the insulated bottle over a 5-hour period, corresponding to the fertilization process in the production of in vitro fertilized eggs of cattle, following the maturation culture process shown in Figure 4A. [Figure 4C] This figure shows the changes in bovine body temperature over a 7-day period, corresponding to the developmental culture process for in vitro fertilized egg production in cattle, following the fertilization process shown in Figure 4B. [Figure 5] This figure shows the transient temperature response characteristics inside the temperature-measuring cylindrical block, which mimics the external shape of the insertable incubator according to the first embodiment, for two different materials, with the block placed inside a constant-temperature water bath set to the average value of bovine body temperature. [Figure 6] Figure 6(a) is a schematic bird's-eye view illustrating an internal insertion tube made of aluminum with a smooth outer surface that was considered by the inventors prior to conceiving the internal insertion type production device according to the first embodiment; Figure 6(b) is a bird's-eye view illustrating the state in which a susceptor is inserted into the internal insertion tube shown in Figure 6(a) to form a spacer; and Figure 6(c) is a bird's-eye view illustrating the state in which a culture vessel is inserted into the hole of the spacer shown in Figure 6(b). [Figure 7] This is a schematic cross-sectional view illustrating the structure of implantable production devices related to Reference Examples 1-8. [Figure 8] Figure 8(a) is a schematic bird's-eye view illustrating a structure in which a transverse groove is formed on the outer circumference of an acrylic resin insert tube, which was considered by the inventors prior to conceiving of the implantable production device according to the first embodiment. Figure 8(b) is a bird's-eye view illustrating a state in which a susceptor is inserted into an acrylic resin aluminum insert tube to form a spacer. Figure 8(c) is a bird's-eye view illustrating a state in which a culture container is inserted into the hole of the spacer in an acrylic resin aluminum insert tube. [Figure 9]Figure 9(a) is a schematic bird's-eye view illustrating an internal insertion cylinder made of aluminum with a smooth outer surface, which was considered by the inventors prior to conceiving the internal insertion type production device according to the first embodiment; Figure 9(b) is a bird's-eye view illustrating a structure in which an aluminum internal insertion cylinder is provided with three vertical grooves; and Figure 9(c) is a bird's-eye view illustrating a structure in which an aluminum internal insertion cylinder is provided with a spiral groove. [Figure 10] This is a bird's-eye view illustrating the structure of a gap setting means (holding device) for setting a gap Vgap used in an insertable culture vessel according to a second modified example of the first embodiment of the present invention. [Figure 11] This is a schematic cross-sectional view illustrating the outline of an insertable culture vessel according to a second modified example of the first embodiment of the present invention. [Modes for carrying out the invention]

[0011] In the following, a first embodiment of the present invention and a modified version thereof will be described illustratively with reference to the drawings. In the drawings referenced in the following first embodiment, its modified version, and second embodiment, identical or similar parts are denoted by identical or similar reference numerals. However, it should be noted that the drawings are schematic, and the relationship between thickness and planar dimensions, the ratio of the sizes of each component, etc., may differ from reality. Therefore, specific thicknesses, dimensions, sizes, etc., should be judged in a more diverse manner, taking into account the essence of the technical idea that can be understood from the following description. Furthermore, it goes without saying that there are parts where the relationships and ratios of dimensions differ between drawings.

[0012] Furthermore, the first embodiment and its modifications, as well as the second embodiment and its modifications, described below, illustrate methods and apparatus used in those methods for realizing the technical concept of the present invention. The technical concept of the present invention does not limit the materials, shapes, structures, arrangements, etc., of the components, or the procedures of the methods to those described below. The technical concept of the present invention is not limited to the contents described in the first embodiment and its modifications, or the second embodiment, and various modifications can be made within the technical scope defined by the claims described in the patent claims. In addition, the definitions of directions such as up and down in the following description are merely for the convenience of explanation and do not limit the technical concept of the present invention. For example, if an object is rotated 90° and observed, "up" and "down" will be converted to "left" and "right" and read accordingly, and if it is rotated 180° and observed, "up" and "down" and "left" and "right" will be reversed and read accordingly.

[0013] (Internal implantable production device according to the first embodiment) The internally insertable production device 1a according to the first embodiment of the present invention comprises, as illustrated in Figure 1A, a substantially cylindrical outer casing 21 that encloses a substantially cylindrical storage cavity inside, and a culture container 10 housed inside the storage cavity of the outer casing 21. As shown in Figure 1B, the cross-sectional structure is shown, with a bottom at one end (left side) of the storage cavity of the outer casing 21, and the outer casing 21 has a no-end recess that constitutes a closed container (storage cavity) with a no-end recess. On the open end (right side) of the outer casing 21 with the no-end recess, a housing lid 23 having a screw portion as shown in Figure 1D is detachably provided, constituting a container with a lid. The outer casing 21 and the housing lid 23 constitute a substantially cylindrical outer casing (21, 23) that encloses a substantially cylindrical storage cavity as a closed space inside. The screw portion is a male screw with a root diameter of d1 and a peak diameter of d2, as shown in Figure 1D(b). On the other hand, since the outer casing 21 has female threads on the open end side, the male threads of the casing cover 23 are screwed into the female threads. As shown in Figures 1A and 1C, two flexible wing portions 25a and 25b are attached to one outer end (the left end) of the outer casing 21 located at the bottom of the storage cavity so as to function as stoppers during insertion (these are not shown in Figure 1B).

[0014] As shown in Figure 1D, a string connection part with a hole of diameter φ is provided at the end of the housing cover. When the blade parts and the string connection part are included, the in-vivo insertion type production tool according to the first embodiment can be defined as a structure having an outer skin part. As shown in Figure 1D(c), the length of the portion of the string connection part in contact with the housing cover is L4. As shown in Figure 1A, a long operation string with a length of about several tens of cm to 1 m is attached to the hole of the string connection part. When inserting the in-vivo insertion type production tool into the vagina of a cow (hereinafter referred to as "cow vagina 9 v "), as shown in Figure 1A, the operation string is always hung out of the cow vagina 9 v so that operation from the outside of the cow vagina 9 v is enabled. As shown in Figure 1B, the in-vivo insertion type production tool according to the first embodiment covers at least a part of the culture vessel in the substantially cylindrical cavity inside the storage cavity of the outer skin housing with a gas layer such as air, and the gas layer forms a thermal buffer sheath 11 a to improve temperature stability, which is a feature of the structure. As will be described later with reference to Table 1, the thermal conductivity of air is 0.0257 (W / (m·k)). In contrast, the thermal conductivity of argon (Ar) gas is 0.016 (W / (m·k)), so from the perspective of thermal conductivity, if Ar gas is used instead of air, the thermal buffer sheath 11 a can have improved heat insulation effect and temperature stability. The thermal buffer sheath 11 a has a thermal buffer function that slows down the change of thermal transient response and improves temperature stability by increasing the thermal time constant τ v that regulates the heat flow between the culture fluid stored in the culture test tube and the in-vivo structure of the cow such as the cow vagina 9 th .

[0015] By configuring the in-vivo insertion type production tool according to the first embodiment as a lidded container with a detachable housing cover, at least a part of the culture vessel is placed in the substantially cylindrical storage cavity inside the outer skin part with the thermal buffer sheath 11 having high temperature stability aCovered with a heat-insulating sheath 11 inside the storage cavity a It can be stored in a sealed state via the casing. The culture container 10 may be shaped to fit the storage cavity of the outer casing (21, 23, 23b, 25a, 25b), but the shape is not limited as long as it can be stored in the storage cavity of the outer casing (21, 23, 23b, 25a, 25b). Cow vagina 9 v From the perspective of inserting it into the body of the cow 9 via and using it repeatedly for a long period of time, Figure 1A shows the surface of the outer skin (21, 23, 23b, 25a, 25b) and the operating string 12 etc. of the cow vagina 9. v It is preferable that the entire component in contact with the device is made of a material such as medical-grade silicone rubber.

[0016] The culture container 10 housed inside the storage cavity of the internally insertable production device 1a according to the first embodiment, as shown in Figures 1B and 1E, consists of a culture test tube 10a that is elongated cylindrical with one end (left end) closed in a rounded bottom shape, and a test tube cap 10b that covers the other open end of the culture test tube 10a and seals the inside of the culture test tube 10a. Inside the culture container 10 is a culture fluid 8, which is a mixture of culture medium containing fertilized or unfertilized eggs and gas. The culture test tube 10a has a holder 24a made of resin or the like, which is hollow cylindrical (perforated cylindrical) as shown in Figure 1E(b), with a gap V around the culture test tube 10a. gap It is held and used as a gap setting means for setting the gap V. In Figure 1B, the gap V gap The volume V = V1 + V2. By the simplified operation of inserting the culture test tube 10a into the holder 24a, which serves as a gap setting means, the longitudinal orientation of the culture test tube 10a and the gap V gap The enclosed positions are automatically determined and housed inside the storage cavities of the outer layer (21, 23, 23b, 25a, 25b), thus improving structural reproducibility and enabling optimization of the entire process of in vitro fertilization embryo production technology.

[0017] In other words, by defining the orientation and position of the culture test tube 10a using the holder 24a, the structure of the internally insertable production device 1a according to the first embodiment can be standardized, thereby transforming the in vitro fertilization embryo production process. Standardization of the structure simplifies the preparation work for embryo transfer using the internally insertable production device 1a, allowing for more stable and easier execution of reproducible and reliable embryo transfer preparation work, thereby increasing the profitability of producers (farmers). Therefore, it contributes to the dissemination of simple in vitro fertilization embryo production technology that farmers can implement, thereby increasing the value of farmers. In particular, by designing the outer diameter of the holder 24a to be approximately close to the inner diameter of the storage cavity in the outer shell (21, 23, 23b, 25a, 25b), a heat buffer sheath 11 made of air or Ar gas surrounds the culture test tube 10a, as shown in Figure 1B. a However, it is possible to adjust the gap to be set in a symmetrical structure with uniform thickness in the radial direction perpendicular to the longitudinal direction. Therefore, a gap V is formed around the culture test tube 10a. gap The inner diameter of the through-hole of the holder 24a, which serves as a gap setting means for setting the gap, is designed to be a hollow cylindrical structure that is approximately close to the outer diameter of the culture test tube 10a. Then, as shown in Figure 1E(b), the culture test tube 10a can be precisely inserted into the through-hole of the holder 24a, and the longitudinal central axis of the culture test tube 10a can be aligned with the longitudinal central axis of the storage cavity. A thermal buffer sheath 11 made of a gas layer a By arranging the elements in a symmetrical structure around the culture test tube 10a, the thermal time constant τ that defines the heat flow is established. th As the temperature of the culture fluid 8 increases, the transient response changes due to heat flow become slower, e Rapid changes are suppressed, and temperature stability is improved.

[0018] The internally inserted production device 1a according to the first embodiment is, as shown in Figure 2, a cow vagina 9 v The culture vessel 10 is inserted into the body of the cow 9 via the T bThe purpose is to culture in vitro fertilized eggs to a stage where they can be transplanted to recipient cows (transplant cows) (blastocyst stage), or to culture unfertilized eggs to a stage where they can be fertilized, by maintaining a temperature of 38-39°C. However, according to the inventors' findings, as shown in Figure 4C described later, the bovine body temperature T b It became clear that there is a circadian rhythm, and it is necessary to consider that it fluctuates depending on the time of day: T b = f(t) ……(1) As is well known, the bovine body temperature T shown in equation (1) is b The fluctuations also depend on changes in barn temperature and the state of estrus. In addition, as shown in Figure 4C, the cow body temperature T b However, the diurnal rhythm, which is higher during the day and lower at night and in the early morning, depends on the cow's sensitivity to thermal factors.

[0019] In other words, the inventors have developed a bovine body temperature T as shown in Figure 4C. b Considering the changes, we have obtained new insights that increasing the thermal conductivity of the internally inserted production device 1a leads to a problem in which the rate of fertilized eggs reaching the blastocyst stage decreases. In other words, the culture fluid 8 and cow vagina 9 v The thermal time constant τ for heat conduction between the body of a cow and other animals. th If the temperature is small, the temperature T of the culture fluid 8 stored in the culture vessel 10 e The changes are T e =g(t)=g * (T b , τ th ) ... (2) The time-dependent variation is close to equation (1). According to the inventors' experiments described later, the thermal time constant τ th If it is small, the temperature T of the culture fluid 8 shown in equation (2) e It was found that the temperature stability deteriorated significantly over time, leading to a decrease in the blastocyst formation rate. Therefore, as shown in Figure 1B, a heat buffer sheath 11 was placed around the culture test tube 10a. a This is provided in a symmetrical structure with a uniform thickness to enhance temperature stability.

[0020] As shown in Figures 1A and 1C, the first blade portion 25a and the second blade portion 25b of the internally insertable production device 1a are identical in shape, and their ends are fixed to the part corresponding to the bottom of the cylinder of the outer casing 21, but they are flexible and can be deformed. As shown in the left side view of Figure 1C(b), the first blade portion 25a and the second blade portion 25b are symmetrical with respect to the central axis extending in the longitudinal direction of the outer casing 21 when unloaded. In the example shown in Figure 1A, it is difficult to discern, but the connection portion of the first blade portion 25a to the outer casing 21 has a structure that rises up over an extremely short distance along the longitudinal direction of the outer casing 21. At the tip of the short connection portion, it bends upward at a predetermined angle of inclination, becoming a blade portion that extends outward (upward in Figure 1C) from the central axis in the longitudinal direction of the outer casing 21.

[0021] Similarly, in the second wing portion 25b, in the example shown in Figure 1C, the connection portion to the outer casing 21 rises up over an extremely short distance along the longitudinal direction of the outer casing 21. At the tip of the short connection portion, it bends downward at an angle opposite to that of the first wing portion 25a, and has a wing portion that extends outward from the central axis in the longitudinal direction of the outer casing 21. In the examples shown in Figures 1A and 1C, the cow vagina 9 v Considering the static position within the body of the cow 9, the initial setting value (unloaded setting value) of the outward bending inclination angle of the first wing portion 25a and the second wing portion 25b is preferably 70 to 90 degrees with respect to the longitudinal central axis of the outer shell housing 21, and more preferably 80 degrees. Also, in the example shown in Figures 1A and 1C, the length L3 of the wing portion of the second wing portion 25b is the cow vagina 9 v Considering the static position within the body of the cow 9, the length is preferably 5 to 7 cm, and more preferably 6.4 to 6.7 cm.

[0022] Since the first wing portion 25a and the second wing portion 25b are made of an elastic (flexible) material such as medical-grade silicone rubber, the inclination angles exemplified in Figures 1A and 1C can be arbitrarily changed. Although not shown in the illustration, the first wing portion 25a and the second wing portion 25b have flexibility that allows them to freely change shape so as to extend parallel to the longitudinal direction of the outer casing 21 (see Figure 9B of Patent Document 2), or so as to allow the wing portion to adhere closely to and sandwich the longitudinal side surface of the outer casing 21 (see Figure 9A of Patent Document 2). When the first wing portion 25a and the second wing portion 25b change shape so as to extend in a manner that sandwiches the side surface of the outer casing 21, in addition to the original bent portion at the boundary between the connection portion to the outer casing 21 and the wing portion, each wing portion will have one bent portion. When the shape changes to extend parallel to the longitudinal direction of the outer casing 21, the bent portions of the first wing portion 25a and the second wing portion 25b are extended without being bent.

[0023] The inclined state shown in Figures 1A and 1C, and the state extending parallel to the longitudinal direction, can be easily changed by the pressure of a human finger. Since the first wing portion 25a and the second wing portion 25b are flexible, they easily return to the state shown in Figures 1A and 1C when the pressure of a human finger is released. The respective wing portions of the first wing portion 25a and the second wing portion 25b may be "flat" in shape as shown in Figures 1A and 1C, or the cross-sectional shape perpendicular to the longitudinal direction may be "fish cake" shaped. When the cross-sectional shapes of the first wing portion 25a and the second wing portion 25b are "fish cake" shaped with a convex shape toward the outside, the cow vagina 9 v After the cow 9 has been left to stand in the body via the phalanx, the endometrium and cow vagina 9 of each feather portion v The surface area of ​​the outer part that comes into contact with the body of the cow 9 is reduced, making it possible to reduce stress on the cow 9.

[0024] From the perspective of reducing the burden on the cow 9, the first wing portion 25a and the second wing portion 25b are extended parallel to each other along the longitudinal direction of the outer shell housing 21, and the extended tip side is the tip in the insertion direction, and the cow vagina 9 vAn internally inserted production device 1a, consisting of an outer shell casing 21 containing a culture container 10, is inserted into the body of the cow 9 via the casing. The internally inserted production device 1a is inserted in a uniform columnar shape extending along the longitudinal direction of the outer shell casing 21, and when the insertion end of the internally inserted production device 1a, i.e., the first wing portion 25a and the second wing portion 25b, reach the vicinity of the cervix, the folded shape of the first wing portion 25a and the second wing portion 25b elastically returns to the inclined state shown in Figures 1A and 1C, etc., and opens. When it returns to the inclined state, the inclined portions of the first wing portion 25a and the second wing portion 25b each become stoppers (movement limiting parts), and the internally inserted production device 1a does not easily enter the cow's vagina 9 v It will not be exposed to the outside. Because it is flexible, at the start of labor, the pressing force from the fetus and secretions from the uterus will increase, causing it to deform into a uniform columnar shape or something close to it that extends in the longitudinal direction, and the internally inserted production device 1a will be inserted into the cow vagina 9 v It is pushed back outwards. The pressing force increases, and the internally inserted production device 1a is pushed out of the cow vagina 9 v When fully exposed to the outside and dropped, it is released from pressure, and the shape of the internally inserted production device 1a elastically returns to the state shown in Figures 1A and 1C.

[0025] The operating cord 12 can be wrapped around, for example, the tail of the cow 9 to prevent the internally inserted production device 1a from falling, and to prevent damage to the internally inserted production device 1a due to falling or subsequent trampling by the cow 9. By using the operating cord 12, the internally inserted production device 1a can be inserted into the uterus or vagina 9 of the cow 9. v This prevents accidents where the device becomes stuck inside the device and allows for easy removal of the implantable production device 1a at any time to observe the culture state. The operating cord 12 is preferably made of medical-grade silicone rubber or a similar material, but the material is not restricted. The operating cord 12 may be a different color from the outer casing (21, 23, 23b, 25a, 25b). The operating cord 12 may be integrally molded with the housing lid 23 or the cord connection part 23a. If the operating cord 12 is integrally molded with the housing lid 23 or the cord connection part 23a, the hole in the cord connection part 23a can be omitted.

[0026] The heat flow between the culture fluid 8 and the cow 9 can be represented by a ladder-shaped distributed multiplier circuit shown in Figure 3, focusing on heat conduction. However, the thermal buffer sheath 11 a When the structure is composed of a gaseous layer such as air, it is important to note that three heat flow mechanisms must be considered, including conductive heat flow, radiant heat flow, and convective heat flow. In Figure 3, the thermal resistance r 10a r is the thermal resistance in the direction normal to the side surface of the culture test tube 10a, and the thermal resistance r 21 r is the thermal resistance in the direction normal to the side surface of the outer casing 21, and the thermal resistance r 24 This is the thermal resistance of the holder 24a perpendicular to the longitudinal central axis of the culture test tube 10a. 10a ,r 21 ,r 24 The thermal resistance r determines the value of such values. th A larger value indicates that heat is less easily transferred. Thermal resistance r th The value of is given by the following equation (3) when the shape is a rod-like shape with a constant cross-sectional area: r th =L / (λ·A)=ψ(L,A) / λ ……(3) In equation (3), L is the length (m) and A is the cross-sectional area (m²). 2 ), λ is the thermal conductivity (W / (m·K)), and ψ(L,A) is the length L (m) and cross-sectional area A (m). 2 It is a shape factor determined by ). Also, thermal resistance r 11a is a thermal buffer sheath 11 a The thermal resistance of the gas layer constituting the culture test tube 10a is perpendicular to the longitudinal central axis, and the heat capacity C 11a This is the thermal buffer sheath 11 between the culture test tube 10a and the holder 24a. a This is the heat capacity of the gas layer that makes up the thermal buffer sheath 11. a The mass of the gas layer that makes up the gas is m 11a Therefore, the specific heat of the gas at constant volume is c v11a As for heat capacity C 11a teeth, C 11a =m 11a ·c v11a ...(4) It can be expressed as follows.

[0027] The mass of the material constituting the holder 24a is m 24 Therefore, the specific heat of the material constituting the holder 24a is c 24 Therefore, the heat capacity C is also inside the holder 24a. 11a This is a possible explanation. However, inside the holder 24a, the thermal resistance r 24 Heat capacity C 11a When the short circuit occurs and the temperature difference ΔT ≈ 0°C perpendicular to the longitudinal central axis of the culture test tube 10a of the holder 24a can be considered, the heat capacity C 11a The contribution of m can be ignored. 11a Since it is small, if we focus only on the flow of conductive heat, the heat capacity C can be calculated from equation (4). 11a The specific heat of a gas at constant volume is c v11a Even if and are relatively large, they are small values. However, the heat capacity C in equation (4) 11a Considering the three heat flow mechanisms, including radiant heat flow and convective heat flow, the effective heat capacity C in equation (4) 11a This becomes larger as determined by static, interdimensional steady-state heat conduction. In particular, as shown in Figure 4C, the cow body temperature T b When the temperature changes in a circadian rhythm, the side of the outer layer (21,23) or the culture vessel 10 that becomes hotter becomes the heat source for the heat rays (far-infrared radiation), and the flow of radiant heat reverses. That is, considering the dynamic changes in the flow of radiant heat, thermal oscillations in which the flow of radiant heat alternately reverses are repeated between the outer layer (21,23) and the culture vessel 10, resulting in a pseudo-resonance state where thermal energy is stored. Therefore, considering the transient phenomena in dynamic heat conduction due to the three heat flow mechanisms, the effective heat capacity C in equation (4) is... 11a The value of increases due to the large mass of the outer shell (21,23) and the heat capacity of the culture vessel 10.

[0028] On the other hand, the outer shape r o , inner diameter r i The shape factor ψ(L,A) in equation (3) when heat passes radially through a cylinder of length L is, ψ(L,A)=ln(r o / r o) / 2πL ……(5) is obtained. From the form factor ψ(L,A) of equation (5), when each of the length of the substantially cylindrical outer shell portions (21, 23) and the length L of the substantially cylindrical culture vessel 10 is greater than the cylindrical thickness t=r o -r o , it is found that the value of the thermal resistance r in equation (3) becomes small. If the holder 24a is made of an acrylic resin and the culture test tube 10a is made of borosilicate glass or Pyrex (registered trademark) glass, in view of the value of the form factor ψ(L,A) of equation (5) and the thermal conductivity λ shown in Table 1, in FIG. 3, r th can be seen to be small. If the holder 24a is made of an acrylic resin and the culture test tube 10a is made of borosilicate glass or Pyrex (registered trademark) glass, in view of the value of the form factor ψ(L,A) of equation (5) and the thermal conductivity λ shown in Table 1, in FIG. 3, r 10a ,r 21 ≪r 11a can be regarded. When this approximation holds, the thermal time constant τ associated with the radial heat propagation of the thermal buffer sheath 11 a is, the thermal time constant τ associated with the radial heat propagation of the thermal buffer sheath 11 th , τ th =C 11a ·r 11a ……(6) can be expressed in a simplified manner. As described above, it is necessary to consider three heat flows when the thermal buffer sheath 11 a is composed of a gas layer such as air. Considering the mechanisms of the three heat flows, the thermal time constant τ of equation (6) th increases, and it can be seen that both the good heat flow property and the good temperature stability of the culture vessel 10 due to the increase of the thermal time constant τ by the thermal buffer sheath 11 a are enhanced, the thermal time constant τ by the thermal buffer sheath 11 th are enhanced.

Table 1

[0029] Actually, inside the culture test tube 10a, where the mass of the culture fluid 8 stored inside the culture test tube 10a is m8 and the specific heat of the culture fluid 8 is c8, C8=m8·c8……(7) Thermal energy is stored due to the heat capacity C8. Therefore, a temperature distribution and thermal delay occur inside the culture fluid 8 along the longitudinal direction (thrust direction) of the culture test tube 10a. When the culture fluid 8 is separated into the liquid phase of the culture medium and the gaseous culture gas phase, a temperature distribution and thermal delay in the thrust direction occur in both the liquid phase and the gaseous culture gas phase. For this reason, the culture fluid 8 contained inside the culture test tube 10a has a thermal time constant τ of equation (6). th The thermal delay in the thrust direction defined by the equation will propagate in a distributed-parameter circuit manner, resulting in a transient response. Therefore, as shown in equation (1), the bovine body temperature T b Even if the temperature changes according to the circadian rhythm, the thermal delay allows the culture fluid 8 to achieve the temperature stability necessary for culturing fertilized or unfertilized eggs, and the bovine body temperature T b The heat flow from there can be effectively utilized.

[0030] (An internally implantable production device relating to the first modified example of the first embodiment) Figure 1B shows an example in which the culture vessel 10 is held near the center by a single holder 24a, and the direction and position of the long axis of the culture vessel 10 are adjusted, but this is merely an example. For example, in the internally inserted production device 1c according to the first modification of the first embodiment of the present invention shown in Figure 1F, a first holder 24a1 and a second holder 24a2, which serve as gap setting means, are arranged opposite each other along the main axis of the long axis of the storage cavity at both ends of the culture vessel 10 in the longitudinal direction. That is, in Figure 1F, there are gaps V at three locations: to the left of the first holder 24a1, around the culture vessel 10 between the first holder 24a1 and the second holder 24a2, and to the right of the second holder 24a2. gap The first holder 24a1 and the second holder 24a2 are positioned opposite each other along the longitudinal main axis of the storage cavity, thereby holding the culture container 10 in a hollow state and creating a gap V gap By setting this, the gap V gap to heat buffer sheath 11 a This configuration enhances temperature stability. As shown in Figure 1F, two first holders 24a1 and second holders 24a2 are placed with a gap V gap If used as a means for setting the gap, the thermal buffer sheath 11 is made of a gas such as air or Ar gas. aHowever, it can be configured to have a symmetrical structure with uniform thickness in the radial direction perpendicular to the longitudinal direction.

[0031] That is, the orientation and position of the culture test tube 10a are defined by using the first holder 24a1 and the second holder 24a2 at both ends, and the heat buffer sheath 11 a By setting the structure symmetrically, it becomes possible to standardize the structure of the implantable production device 1c according to the first modification of the first embodiment. This standardization simplifies the preparation work for embryo transfer using the implantable production device 1c, and allows for more stable and reliable execution of tasks such as maturation culture and developmental culture. Specifically, in the implantable production device 1c according to the first modification of the first embodiment shown in Figure 1F, a first holder 24a1 is positioned near the bottom on the left side of the storage cavity of the outer shell (21, 23), and a second holder 24a2 having an inner diameter matching the outer diameter of the test tube cap 10b is installed on the right side, which is the side of the test tube cap 10b, and a heat buffer sheath 11 is placed around the culture container 10. a It is embedded in the gap. According to the internally inserted production device 1c of the first modified embodiment, the bovine body temperature T b While ensuring good heat flow from, the thermal buffer sheath 11 a By providing this, the thermal time constant τ in the heat flow th By increasing this, good temperature stability of the culture vessel 10 can be achieved. Note that in Figure 1F, the first holder 24a1 and the second holder 24a2 are shown as hollow cylinders with through holes, but this is merely an example. For example, the first holder 24a1 may be a convex-to-left concave shape, and the second holder 24a2 may be a convex-to-right concave shape.

[0032] As explained in Patent Document 2, the in vitro fertilization of a cow 9 involves three steps prior to the embryo transfer to the recipient cow: (a) a maturation culture step requiring 1 day, (b) a fertilization step requiring 6 hours, and (c) a developmental culture step requiring 7 days. Figure 4A shows the cow's body temperature T over a 24-hour period corresponding to the time required for the maturation culture step (a) of the three steps. bThis shows the change over time. The dashed line in Figure 4A shows the average value of 38.2°C, but it also shows that there is a temperature fluctuation of 39.0°C to 37.8°C over 24 hours. The temperature measurement corresponding to the maturation culture process in Figure 4A is achieved by housing a plate-shaped temperature sensor with data storage function inside the outer skin (21,23) of the internally inserted production device 1c according to the first modified example of the first embodiment, and placing this outer skin (21,23) into the cow vagina 9 v It is placed inside the body of a cow 9, and after 24 hours, the cow vagina 9 v The outer skin (21,23) is removed from the body of a cow 9, and a temperature sensor is connected to a computer to output temperature data to the computer. The plate-shaped temperature sensor is positioned near the bottom of the left end of the housing cavity of the outer skin (21,23), whose cross-sectional structure is shown in Figure 1F, with its main surface perpendicular to the longitudinal direction of the housing cavity. The structure of the outer skin (21,23) housing the temperature sensor is the same as the cross-sectional structure shown in Figure 1F, but the culture container 10 is also housed in the housing cavity of the outer skin (21,23) such that the end face of the culture container 10 is adjacent to the main surface of the plate-shaped temperature sensor. As shown in Figure 1F, the first holder 24a1 and the second holder 24a2 are positioned opposite each other along the longitudinal direction of the housing cavity at both ends of the culture container 10 in the longitudinal direction of the housing cavity, holding the culture container 10 in a hollow state, and a heat buffer sheath 11 made of a gas layer a This configuration enhances temperature stability. Specifically, in the measurement shown in Figure 4A, the first holder 24a1 is placed near the bottom left of the storage cavity, near a plate-shaped temperature sensor whose main surface is perpendicular to the longitudinal direction of the storage cavity, and the second holder 24a2 is placed on the right side, which is the side of the test tube cap 10b.

[0033] On the other hand, the measurements shown in the graph in Figure 4B correspond to the fertilization process, one of the three processes required for in vitro fertilization egg production, and represent 5 hours out of the required 6 hours. Unlike the temperature measurements corresponding to the maturation culture process in Figure 4A, the temperature data for the water was obtained when 39°C water was placed in a thermos bottle, the culture container 10 was inserted vertically with the culture test tube 10a facing downwards, and the test tube cap 10b was positioned higher than the top surface of the 39°C water. The dashed line in Figure 4B shows the average value of 39.1°C, but indicates that there is a temperature fluctuation within the range of 39.5°C to 38.7°C over the 5 hours. In the temperature measurement under the conditions corresponding to the fertilization process shown in Figure 4B, the temperature sensor with data storage function is located directly below the bottom of the culture test tube 10a at the bottom of the thermos bottle. The graph for the fertilization process shown in Figure 4B represents the temperature measurement data for 5 hours of the corresponding water temperature during the 6-hour fertilization process. The water in the thermos bottle is replaced 3 hours after the start of the fertilization process, which is why the temperature rises after 3 hours.

[0034] The graph shown in Figure 4C shows the bovine body temperature T corresponding to (c) the developmental culture process (culture period of 7 days), one of the three processes required for in vitro fertilization egg production. b This is measurement data showing the change over time. Similar to the maturation culture process in Figure 4A, the data was obtained by placing a temperature sensor with data storage function near the bottom of the left end of the storage cavity, whose cross-sectional structure is shown in Figure 1B. That is, in the temperature measurement corresponding to the developmental culture process, the outer skin portion (21,23) where the temperature sensor was placed was the cow vagina 9 v It is placed inside the body of the cow 9 for 7 days via the cow vagina 9, and after 7 days have elapsed, v The outer skin (21,23) was removed from the body of the cows (9), and data was obtained by connecting a temperature sensor to a computer. From the time course shown in Figure 4C, the cow body temperature T bThis shows that temperature fluctuations in a circadian rhythm, as modeled by equation (1), occur over time. Specifically, Figure 4C shows that the temperature fluctuates in a circadian rhythm of approximately 2°C, from a minimum of 37.7°C to a maximum of 39.6°C, but as shown by the dashed line, the average temperature fluctuation corresponding to the 7-day culture period is 38.5°C. Many carbon dioxide culture devices have a space layer around the inner tank, and temperature stability is ensured by a water jacket filled with water or an air jacket filled with air. However, the data shown in Figure 4C indicates that if the heat conductivity of the implantable production device is too good, the cow body temperature T b It can be seen that fluctuations in this directly affect the culture fluid 8, worsening the culture environment.

[0035] Figure 5 shows the temperature as the bovine body temperature T b A constant-temperature water bath was prepared with an average temperature of 38.5°C. A cylindrical block for temperature measurement, which mimics the external shape of the internally insertable production device according to the first embodiment, was placed in this constant-temperature water bath, and the internal temperature (°C) of the cylindrical block for temperature measurement was measured. However, the inside of the cylindrical block for temperature measurement does not have a cavity for housing the culture container 10 as shown in Figure 7, but is a uniformly filled cylindrical structure. The outer surface is a uniform cylindrical structure, but it is configured to be divided into two parts near the center by the fitting of a cylinder with a male thread and a cylinder with a female thread. The main surface of a plate-shaped temperature sensor is in close contact with the end surface of the cylindrical block at the end of the cylinder at this division point. The temperature sensor has a data storage function similar to the temperature sensor in the measurements in Figures 4A and 4C. That is, the plate-shaped temperature sensor is placed in close contact with the end surface of the divided part of the cylindrical block, with its main surface perpendicular to the longitudinal direction of the cylindrical block, and the internal temperature of the cylindrical block is measured.

[0036] The cylindrical block used for temperature measurement in Figure 5 is an Al spacer (11) related to Reference Examples 1 and 2 described later. b1 ,11 b2 A structure having ) and an acrylic resin spacer (11) according to Reference Example 8. c1 ,11 c2It can be approximated to a structure having ). However, the cylindrical block for temperature measurement does not have a cavity for housing the culture vessel 10 and is a uniform block. The horizontal axis of Figure 5 shows the elapsed time (h) until it reaches 38.5°C after being placed in the constant temperature water bath, and the vertical axis shows the internal temperature (°C) of the cylindrical block for temperature measurement that is the subject of measurement. The time it took for the internal temperature of the aluminum cylindrical block for temperature measurement, shown by the dashed line, to reach 38.5°C was approximately 34 minutes. In contrast, the time it took for the internal temperature of the acrylic resin cylindrical block for temperature measurement, shown by the solid line, to reach 38.5°C was approximately 1 hour and 12 minutes.

[0037] (Method for preparing fertilized egg for transfer according to the first modified example of the first embodiment) By using the internally implantable production device 1c according to the first modification of the first embodiment, the three steps necessary for in vitro fertilization of cows 9—maturation culture, fertilization, and developmental culture—can be carried out without using a carbon dioxide culture device, and preparation for the fertilized egg transfer step to the recipient cow can be completed. According to the method for preparing for fertilized egg transfer using the internally implantable production device 1c according to the first modification of the first embodiment, a heat buffering sheath 11 is placed inside the internally implantable production device 1c. a By providing the thermal buffer sheath 11 a The thermal time constant τ th By increasing the temperature, good temperature stability of the culture vessel 10 is achieved, and a fertilized egg production environment equivalent to that of an expensive carbon dioxide culture device is created, using the bovine body temperature T b The heat flow from there prepares the area for the embryo transfer process.

[0038] First, the cow's body temperature T bIn the maturation culture process using this method, for example, an ultrasound probe is inserted into the vagina to collect eggs transvaginally (OPU). The collected immature fertilized eggs are then transferred to a culture test tube 10a along with a culture medium for in vitro maturation. The open end of the culture test tube 10a is then closed with a test tube cap 10b to prepare the culture vessel 10 for use in the maturation culture process. For example, to enable in vitro maturation of immature oocytes with a gas phase composition similar to the internal environment of a cow (e.g., 5% carbon dioxide, 95% air), the gas inside the culture vessel 10 is replaced using a gas phase injector such as a syringe, thereby forming and filling the culture fluid 8 inside the culture test tube 10a. Subsequently, the culture vessel 10 is placed inside the storage cavity of the outer shell (21, 23, 23b, 25a, 25b) via the first holder 24a1 and the second holder 24a2 as gap setting means, so that it is in a predetermined orientation and position, and a gap V is left around the culture vessel 10. gap Set the gap V by the gap setting means. gap By setting this, the gap V around the culture vessel 10 gap A thermal buffer sheath 11 made of a gas such as air or Ar gas. a The internally insertable production device 1c for the maturation process is constructed with the embedded material. The internally insertable production device 1c is then inserted into the bovine vagina 9 v Insert into the body, and heat the cow's body temperature T b Using this method, as shown in Figure 4A, a 24-hour maturation culture process is performed as the required maturation period, allowing the collected immature eggs to undergo meiosis to the stage of secondary oocytes capable of fertilization, and thus undergo in vitro maturation.

[0039] After a 24-hour maturation culture process, the embryo matures in vitro to a stage where it can be fertilized, and then the internally inserted production device 1c is inserted into the bovine vagina 9 vThe eggs are removed from the body and the process moves to in vitro fertilization without the use of a carbon dioxide culture device. Specifically, in the fertilization process, the housing lid 23 of the outer casing (21, 23, 23b, 25a, 25b) of the removed internally inserted production device 1c is removed, and the culture test tube 10a is removed from inside the outer casing (21, 23, 23b, 25a, 25b). Furthermore, the test tube cap 10b of the culture test tube 10a is removed, and eggs that have reached the stage where they can be fertilized are collected from inside the culture test tube 10a and in vitro fertilization is performed. The in vitro fertilization process is carried out in an environment that is close to the internal environment of the cow 9, such as 5% carbon dioxide and 95% air. In the fertilization process, eggs are transferred to culture test tube 10a containing a sperm suspension, and culture test tube 10a is placed in a thermos bottle containing 39°C warm water. Fertilization is then carried out by sprinkling sperm onto the eggs over a period of 6 hours, as shown in Figure 4B, to produce fertilized eggs.

[0040] The fertilized eggs produced during the 6-hour insemination process are placed in culture test tube 10a containing culture medium. Then, a test tube cap 10b is placed over culture test tube 10a to prepare the culture vessel 10 to be used in the developmental culture process, and the bovine body temperature T b The process moves to preparation for the developmental culture step using the following: For example, using a gas phase injector such as a syringe, the gas phase inside the culture container 10 to be used in the developmental culture step is replaced to create a culture gas phase composition similar to the internal environment of a cow 9 by using 5% carbon dioxide, 5% oxygen, and 90% nitrogen, thereby forming and filling the inside of the culture container 10 with culture fluid 8. Subsequently, the culture container 10 is placed inside the storage cavity of the outer shell (21, 23, 23b, 25a, 25b) via the first holder 24a1 and the second holder 24a2 as gap setting means, so as to be in a predetermined orientation and position. The gap setting means create a gap V around the culture container 10. gap This gap V is set. gap A thermal buffer sheath 11 made of a gas such as air or Ar gas. a The implanted material constitutes an internally insertable production device 1c for the developmental culture process. Then, the internally insertable production device 1c is inserted into the bovine vagina 9 v It is left inside, and the cow's body temperature T b Using this, as shown in Figure 4C, a developmental culture process is carried out for 7 days as the required developmental culture period. After the 7-day developmental culture process, the internally inserted production device 1c is inserted into the bovine vagina 9 vRemove from the inside. Remove the housing lid 23 from the outer casing (21, 23, 23b, 25a, 25b) of the removed internally inserted production device 1c, and remove the culture test tube 10a from inside the outer casing (21, 23, 23b, 25a, 25b). Furthermore, remove the test tube cap 10b from the culture test tube 10a, collect the oocytes from inside the culture test tube 10a, and check using a stereomicroscope or the like whether or not fertilized eggs suitable for embryo transfer have been produced. If they are suitable for transfer, proceed to the embryo transfer process to the next recipient cow.

[0041] <Reference examples 1~8> Before conceiving the structure of the implantable production device 1a according to the first embodiment as illustrated in Figure 1B (or the implantable production device 1c according to the first modification of the first embodiment as illustrated in Figure 1F), the inventors investigated and prototyped the structures of implantable production devices according to Reference Examples 1 to 8, as shown in Table 2. Specifically, egg production demonstration tests were conducted using the implantable production devices according to Reference Examples 1 to 8, and the test data were compared and evaluated with the test data of Examples 1 to 3 and the prior art 1 to 3. Table 2 also shows the weight and height of the test cows. [Table 2]

[0042] As already mentioned, the in vitro fertilization of bovine embryos involves three steps: (a) maturation culture, (b) fertilization, and (c) developmental culture. Reference examples 4, 6, and 8 only include the developmental culture step of the three steps, and the bovine body temperature T b Using this method, the maturation culture process and fertilization process are egg production demonstration tests using a carbon dioxide culture device. Reference example 2 is a cow vagina 9 during the maturation culture process. v The test has been stopped because the internally inserted production device fell out from inside. At the start of the demonstration test, cow vagina 9 vThe rupture detection housing repeatedly detached from the inside. Therefore, in Reference Examples 3-8 and Examples 1-3, two umbrella-shaped stoppers made from commercially available suction cups were added to the outer casing of the internally inserted production device near the center, spaced apart at positions that divide the longitudinal direction of the outer casing into three equal parts, to prevent the internally inserted production device from detaching (the umbrella-shaped stoppers are not shown in Figure 1F). In the egg production demonstration tests of Reference Examples 1, 3, 5, 7 and Examples 1-3, cow vagina 9 was used in the culture process and developmental culture process. v The procedure involves inserting an internally implantable production device into the cow's vagina 9. v The procedure is performed by extracting the material from within, but a carbon dioxide culture device is not used.

[0043] In the egg production demonstration tests according to Examples 1 to 3 in Table 2, the structure of the internally inserted production device 1c according to the first modification of the first embodiment shown in Figure 1F is adopted. That is, in the internally inserted production device 1c according to Examples 1 to 3, the first holder 24a1 and the second holder 24a2 are positioned opposite each other and spaced apart along the main axis which is the longitudinal direction of the storage cavity to hold the culture container 10. And, as shown in Figure 1F, a heat buffer sheath 11 made of air a The temperature stability is enhanced by configuring the region around the culture vessel 10 between the first holder 24a1 and the second holder 24a2. In the rightmost column of Table 2, the denominator of the "Contents" column showing the development rate is the number of fertilized eggs introduced, and the numerator is the number of fertilized eggs that reached the blastocyst stage, which is suitable for transplantation into the recipient cow. Also, the "%" column showing the development rate in Table 2 is the percentage obtained by dividing the numerator of the "Contents" column by the denominator. For example, in the data display for the row of Example 1, the development rate is 4 ÷ 11 = 36.4%. The data for Conventional Technology 1 to 3 in Table 2 shows the results of egg production demonstration tests using a carbon dioxide culture device.

[0044] The internally implantable production devices shown in Table 2, Reference Examples 1-8, have spacers 11 around the culture vessel 10 as shown in Figure 7. x1 and spacer cap 11 x2 The inner container covered with (10a, 10b, 11 x1 ,11 x2The material is filled into the storage cavity of the outer skin portion (21, 23, 23b, 25a, 25b), which has a structure similar to that shown in Figures 1A, 1B, and 1F, so as to be in close contact with the inner wall. The inventors have developed a method for determining bovine body temperature T b As a thermal design that efficiently utilizes 38~39°C, spacer 11 x1 and spacer cap 11 x2 As a constituent material, aluminum (Al), which has high thermal conductivity and low manufacturing cost, was selected, and an internal container (10a, 10b, 11) made of Al was used. x1 ,11 x2 The following were considered as reference examples 1 to 7. The thermal conductivity of Al at 20°C is 204 (W / (m·K)), as shown in Table 3. [Table 3]

[0045] Considering the thermal conductivity of the materials shown in Table 3, the spacer 11 of the internal container used in the implantable production device according to Reference Example 8 x1 and spacer cap 11 x2 For comparison, acrylic resin was used as a constituent material. As can be seen from Table 3, the thermal conductivity of acrylic resin at 20°C is about three orders of magnitude smaller than that of Al. That is, as reference example 8, a structure as shown in Figure 8(b) was manufactured using a 3D printer, and spacer 11 x1 and spacer cap 11 x2 The effect of the fertilized egg culture environment, due to its thermal conductivity, on the blastocyst formation rate was examined by comparing data from the egg production demonstration test shown in Table 2. Figure 8(a) shows the inner tube 11 manufactured using a 3D printer. c1 This is the structure of the inner tube 11 c1 In contrast, as shown in Figure 8(b), susceptor 11 c2 The two-part structure with the spacer (11 c1 ,11 c2 ) is formed. Then, as shown in Figure 8(c), susceptor 11 c2 The culture test tube 10a is fitted so as to be in close contact with the inner wall. In Figure 8(c), a test tube cap 10b is placed over the open end of the culture test tube 10a.

[0046] Similar to the structure shown in Figure 1F, the left end of the storage cavity of the outer casing (21, 23) of the internally insertable production device shown in Reference Examples 1-8 in Table 2 has a bottom, forming a dead-end space, and the internal container (10a, 10b, 11) shown in Figure 7 x1 ,11 x2 Fill with ). As already explained, Figures 1B and 1F are model diagrams, and in reality, as shown in Figure 1A, the left side of the outer casing 21 is provided with wing parts 25a and 25b, and the operating string 12 is connected to the housing lid 23 on the right side of the outer casing 21 via a string connection part 23a, etc. That is, in the exemplary structure of Figure 1F, a housing lid 23 having a screw part similar to the structure shown in Figure 1D is detachably provided on the open end side on the right side of the outer casing 21, forming a container with a lid. Internal container (10a, 10b, 11 x1 ,11 x2 ) is a roughly cylindrical spacer 11 as shown in Figure 7. x1 and a roughly cylindrical spacer cap 11 x2 However, it is a roughly cylindrical structure in which the outer diameters are shared, and a culture container 10 consisting of a culture test tube 10a and a test tube cap 10b is housed inside the connecting structure.

[0047] As shown in Figure 7, a roughly cylindrical spacer 11 is formed to create a roughly cylindrical body with a common outer diameter. x1 The right end, which is the open end, has a male thread cut into it, and spacer 11 x1 A roughly cylindrical spacer cap 11 having the same outer diameter as the other. x2 It is fitted into the female thread provided in Figure 7. x1 An example of an integrated structure is shown, but as illustrated in Figure 8(b), the inner cylinder 11 is in contact with the inner wall of the outer shell housing 21. c1 and the inner tube 11 c1 Susceptor 11 fitted into the inner diameter side c2 A two-part assembly structure with a stepped section is also acceptable. Susceptor 11 c2 It has a through-hole with an inner diameter close to the outer diameter of the culture test tube 10a. In the case of the assembly structure illustrated in Figure 8(b), the susceptor 11 c2 Internal tube 11c1 A male thread is cut into the stepped portion exposed from the surface, corresponding to the structure in Figure 7, and a spacer cap 11 is attached in the same way as in Figure 7. x2 The female thread provided in the susceptor 11 c2 It is fitted onto the male screw. In any case, as shown in Figure 7, spacer 11 x1 and spacer cap 11 x2 Then, the culture vessel 10 is completely covered with a heat conductor, and a roughly cylindrical inner container (10a, 10b, 11 x1 ,11 x2 It constitutes ).

[0048] The internal containers shown in Figure 7 (10a, 10b, 11 x1 ,11 x2 Spacer 11 located on the surface side of ) x1 and spacer cap 11 x2 This consists of a culture fluid 8 contained in a culture test tube 10a and a cow vagina 9. v It has a heat conduction promoting function that facilitates the flow of heat between the internal structure of the cow 9 and other parts. An internal container (10a, 10b, 11) used by being embedded inside the internally inserted production device according to Reference Examples 1 and 2 shown in Table 2. x1 ,11 x2 As shown in Figure 6(b), the spacer (11) has a uniform and smooth outer surface. b1 ,11 b2 ) is used. Figure 6(a) shows spacer (11 b1 ,11 b2 ) has an inner cylinder 11 having a uniform and smooth outer surface b1 The structure is such that the inner cylinder 11 has a smooth outer surface and is inserted so as to be in contact with the inner wall of the outer shell housing 21. b1 In contrast, as shown in Figure 6(b), susceptor 11 b2 Insert the internal containers (10a, 10b, 11) according to Reference Examples 1 and 2. x1 ,11 x2 ) As shown in Figure 6(c), susceptor 11 b2 The culture test tube 10a is inserted so as to be in close contact with the inner wall, and the test tube cap 10b is placed over the open end of the culture test tube 10a.

[0049] Similar to the example in Figure 8(b), in the case of the assembly structure shown in Figure 6(b), susceptor 11 b2 Internal tube 11 b1 The outer surface of the exposed (protruding) part is threaded with a male screw, and the spacer cap 11 x2 The female thread provided in the susceptor 11 b2 It is fitted onto the male screw. The inner diameter of the medical-grade silicone rubber outer casing 21 is 29 mm. In the reference examples 1 and 2 shown in Table 2, the inner tube 11 b1 and spacer cap 11 x2 (See Figure 7.) The outer diameter is 29 mm, and the inner diameter of the outer shell 21 and the inner cylinder 11 b1 and spacer cap 11 x2 The design was intended to have no gap between the outer diameter and the inner diameter of the inner cylinder 11, in order to improve thermal conductivity. b1 and spacer cap 11 x2 If the design is such that there is no gap between the outer diameter and the inner container (10a, 10b, 11) has a smooth outer surface as shown in Figures 6(a) to (c), the outer shell housing 21 b1 ,11 b2 ,11 x2 It was found that removing the acrylic resin inner tube 11 used in the internal implantable production device shown in Reference Example 8 in Table 2 c1 However, as shown in Figures 8(a) to (c), it has a uniform and smooth outer surface, which similarly makes it difficult to remove.

[0050] Internal containers related to Reference Example 2 (10a, 10b, 11 b1 ,11 b2 ,11 x2 Considering the inconvenience of the detachment of the ) the internal containers relating to Reference Examples 3 to 5 are as shown in Figure 9(b), the inner tube 11 e1 A structure in which three longitudinal grooves extending in the longitudinal direction of the culture test tube 10a are provided on the outer circumference (surface) was compared and examined using data from an egg production demonstration test. In Figure 9(b), the inner tube 11 e1 Susceptor 11 that is inserted into e2 The illustration of the inner tube 11 is omitted. e1By providing three vertical grooves on the outer circumference, it can be predicted that this will affect the heat flow between the culture fluid 8 contained in the culture test tube 10a and the internal structure of the cow 9. Therefore, the inner tube 11 e1 The impact of the thermal issues caused by the three longitudinal grooves on the outer circumference of the fertilized egg culture environment on the blastocyst formation rate was also examined by comparing data from egg production demonstration tests.

[0051] Furthermore, the internal containers related to Reference Examples 5-7 (10a, 10b, 11 f1 ,11 f2 ,11 x2 As shown in Figure 9(c), the inner tube 11 f1 The structure has spiral grooves on its outer circumference (surface), and the effect of the fertilized egg culture environment, caused by thermal conductivity, on the blastocyst formation rate was compared and examined using data from the egg production demonstration test shown in Table 2. In Figure 9(c), as in Figures 9(a) and (b), the inner tube 11 f1 Susceptor 11 that is inserted into f2 The illustration is omitted.

[0052] As shown in Table 2, the aluminum insert 11 b1 ,11 e1 ,11 f1 Results of egg production demonstration tests using internally insertable production devices according to Reference Examples 1-7, which use an internal container made of the above, and the results of the internal insertion tube 11 made of acrylic c1In most egg production demonstration tests using the implantable production device according to Reference Example 8, which uses an internal container composed of the above, the incidence rate was 0%. Exceptionally, an incidence rate of 8.3% was obtained in the egg production demonstration test using the implantable production device according to Reference Example 4, and 16.7% in the egg production demonstration test using the implantable production device according to Reference Example 6. In contrast, the incidence rate in the egg production demonstration test using the implantable production device according to Example 1 was 36.4%, the incidence rate in the egg production demonstration test using the implantable production device according to Example 2 was 18.8%, and the incidence rate in the egg production demonstration test using the implantable production device according to Example 3 was 27.3%. The average incidence rate in the egg production demonstration tests using the implantable production device 1c according to the first modification of the first embodiment according to Examples 1 to 3 is 10 / 38 = 26.3%. On the other hand, the average incidence rate in the egg production demonstration tests using expensive carbon dioxide culture devices related to Conventional Technology 1-3 was 8 / 20 = 40.03%, indicating that the egg production demonstration tests using implantable production devices related to Examples 1-3 yielded results close to the incidence rate of the conventional technology using expensive carbon dioxide culture devices.

[0053] The data from the egg production demonstration test shown in Table 2 indicates that the culture environment for in vitro fertilized eggs from 9 cows was the bovine body temperature T b It is susceptible to changes over time, as shown in Figure 7, the internal container (10a, 10b, 11 x1 ,11 x2 This indicates that there are limitations to technologies that rely on the thermal conductivity of acrylic resin. As can be seen from Table 3, the thermal conductivity of air is an order of magnitude smaller than that of acrylic resin. This means that air is difficult to heat up, but once heated, it has a high heat retention effect. According to the data from the egg production demonstration test shown in Table 2, a heat buffer sheath 11 made of air surrounds the culture test tube 10a. a By adopting the structures of Examples 1 to 3, which include the provision of a bovine body temperature T b Without being affected by fluctuations, the cow's body temperature is almost T b It can be seen that temperature stability can be achieved by keeping culture test tube 10a warm at a constant value centered around the average value.

[0054] The comparison between Reference Examples 1-8 and Examples 1-3 shown in Table 2 is based on the bovine body temperature T bTo produce in vitro fertilized eggs from cows using this method, the cow's body temperature is approximately T b This indicates that it is necessary to maintain the temperature of the culture fluid 8 in an environment with small and gradual fluctuations centered around the average value. The culture fluid 8 for producing in vitro fertilized eggs has a thermal time constant τ th Temperature stability is required in environments with large thermal transient response speeds and slow thermal transient response speeds. In order to design an in vitro fertilized egg production environment with a small temperature fluctuation range and gradual temperature fluctuations, a thermal buffer sheath 11 as shown in Figure 1F is used. a The comparison results in Table 2 indicate that it is necessary to set the thermal resistance r around the culture test tube 10a and the test tube cap 10b. 11a or heat capacity C 11a The thermal time constant τ shown in equation (6) is given by equation (6). th Determine the thermal time constant τ shown in equation (6). th However, since it determines the thermal transient response rate, the cow's body temperature T b This affects the heat flow and temperature stability from the internally implantable production device to the culture test tube 10a, etc.

[0055] Since the thermal transient response rate is important for the production of in vitro fertilized eggs from cattle 9, thermal design is necessary for the in-vivo implantable production device according to the first embodiment and its first modified example. As shown in Figure 7, spacer 11 x1 Spacer cap 11 x2 When tightening, spacer 11 x1 The internally insertable production device is designed so that, as shown in Figure 7, there is space inside to accommodate the culture test tube 10a and the test tube cap 10b. The ladder-shaped distribution multiplier circuit shown in Figure 3 is connected to the bovine body temperature T outside the internal container. b This indicates that when considering the heat flow propagating from the inner container to the culture test tube 10a, it is necessary to consider the components of the heat flow propagating radially through the culture test tube 10a.

[0056] However, in Table 4 below, the internal containers of Reference Examples 1 to 8 are simplified by assuming a hypothetical equivalent cylinder with uniform external dimensions and filled internal storage space, and the thermal resistance of the heat flow propagating in the longitudinal direction (axial direction) of this hypothetical equivalent cylinder is evaluated. [Table 4]

[0057] When evaluating the thermal resistance in Table 4, the internal containers (10a, 10b, 11) related to Reference Examples 1 to 8 shown in Figure 7 should be used. x1 ,11 x2 The external dimensions of the spacer 11 are assumed to be a cylinder with a diameter of 29 mm and a length of 103 mm. That is, the spacer 11 prototyped using aluminum material or acrylic resin material. x1 and spacer cap 11 x2 For a hypothetical equivalent cylinder with uniform external dimensions, the heat flow propagating axially through the equivalent cylinder was considered, and the evaluation in Table 4 was performed. However, in Table 4, the internal container (10a, 10b, 11) shown in Figure 7 is used. x1 ,11 x2 Assuming that an equivalent cylinder with a diameter of 29 mm and a length of 103 mm, equivalent to the outer dimensions of (), is completely filled without gaps with thermal conductive materials such as Al, acrylic resin, and air, the thermal resistance r th We are calculating the (kW) value.

[0058] In equation (3), the L of the hypothetical equivalent cylinder is 1.03 × 10⁻¹⁰. -1 m, A = π × (1.45 × 10 -2 ) 2 = 6.61 × 10 -4 m 2 Using the thermal conductivity of the constituent materials shown in Table 3, the thermal resistance related to the axial heat flow of a hypothetical equivalent cylinder can be obtained as shown in Table 4. Although the heat flow differs from the calculation results shown in Table 4, when the constituent materials around the culture test tube 10a are Al and air, the thermal resistance r th It can be estimated that the (K / W) value differs by about 5 orders of magnitude. The thermal resistance perpendicular to the axial direction of the equivalent cylinder is such that the value of L / A, which is the shape factor ψ(L,A) in equation (3), is smaller than the calculated value shown in Table 4, so the thermal resistance r for Al and airth The difference will become smaller.

[0059] When producing in vitro fertilized eggs from cattle 9 using a conventional carbon dioxide culture device, an initial cost of approximately 500,000 to 5,000,000 yen is required for the installation of the carbon dioxide culture device. In contrast, according to the embryo transfer preparation method employing the internally inserted production device 1a or the internally inserted production device 1c according to the first embodiment, the cattle body temperature T b Because this method is used, the initial cost of introducing equipment for preparing to transplant fertilized eggs into recipient cows is eliminated. Therefore, the method for preparing fertilized egg transplantation using the internally insertable production device 1a according to the first embodiment or the internally insertable production device 1c according to the first modified example makes it possible to reduce the initial cost of introducing equipment for preparing fertilized egg transplantation.

[0060] When using a conventional carbon dioxide culture apparatus, it is possible to produce in vitro fertilized eggs of cattle 9 indoors through three processes: maturation culture (required time: 1 day), fertilization (required time: 6 hours), and developmental culture (required time: 7 days). However, in the embryo transfer preparation method using the internally inserted production device 1a according to the first embodiment or the internally inserted production device 1c according to the first modified example, movement to the barn is required, and the cow's vagina 9 v Because the insertion and removal of the internally implantable production devices 1a and 1c into and out of the cow 9 via the device increases the effort required to prepare for the transplantation of in vitro fertilized eggs into recipient cows. Until now, the preparation process for transplantation of in vitro fertilized eggs in cows 9 using a carbon dioxide culture device could only be performed by veterinarians. According to the method for preparing for embryo transplantation using the internally implantable production device 1a or the internally implantable production device 1c according to the first embodiment, it becomes possible to prepare for transplantation of in vitro fertilized eggs into recipient cows even in cows 9 owned by producers (farmers), thereby reducing costs for producers and enabling the widespread adoption of a simple embryo transplantation technology.

[0061] (An internally implantable production device relating to a second modified example of the first embodiment) Similar to the implantable production device 1a according to the first embodiment, the implantable production device 1b according to the second modification of the first embodiment of the present invention comprises, as illustrated in Figure 11, a substantially cylindrical outer shell (21, 23) containing a substantially cylindrical storage cavity inside, and a culture container 10 housed inside the storage cavity of the outer shell (21, 23) via a gap setting means. The outer shell (21, 23) is composed of an outer shell housing 21 and a housing lid 23, and the culture container 10 is composed of a culture test tube 10a and a test tube cap 10b. However, unlike the implantable production device 1a according to the first embodiment, the implantable production device 1b according to the second modification of the first embodiment holds the culture test tube 10a using a holder 24b made of a hollow cylindrical resin or the like with four vertical grooves 241, 242, 243, 244 on its outer circumference as shown in Figure 10, as a gap setting means. The holder 24b, acting as a gap setting means, determines the longitudinal orientation of the culture test tube 10a and its position inside the storage cavity, and sets the gap around the culture container 10 according to the thermal time constant τ th Large heat buffering sheath 11 a It is filled with this material to improve temperature stability.

[0062] As shown in Figure 10, the outer circumference of the holder 24b of the internally insertable production device 1b according to the second modified example of the first embodiment has longitudinal grooves 241, 242, 243, and 244. Focusing on the locations where the longitudinal grooves 241, 242, 243, and 244 exist, a thermal buffer sheath 11 is also present between the holder 24b and the outer skin (21, 23). a It can be seen that this can be set. Figure 11 shows a cross-section cut at the cross-section C of Figure 10, and if we observe the cross-sectional structure of the internally inserted production device 1b along the cross-section C so as to include the cutting line IX1-IX1, we can see that there is a thermal buffer sheath 11 with a volume ΔV between the holder 24b and the outer skin (21,23). a This setting contributes to further improving temperature stability. Therefore, a gap V is set around the culture vessel 10. gap The volume V is expanded to volume V = V1 + V2 + ΔV in the internally inserted production device 1b according to the second modification of the first embodiment, compared to the case shown in Figure 1B. The orientation and position of the culture test tube 10a are defined using the holder 24b, and the expanded volume V = V1 + V2 + ΔV of the thermal buffer sheath 11 aBy setting the structure symmetrically, it becomes possible to standardize the structure of the internally implantable production device 1b according to the second modification of the first embodiment. Standardization of the structure simplifies the work of preparing fertilized egg transfer using the internally implantable production device 1b, and allows for more stable and reliable execution of operations such as maturation culture and developmental culture, thereby contributing to the dissemination of a simple in vitro fertilized egg production technology that can be implemented by producers (farmers).

[0063] The structure of the retainer 24b shown in Figure 10 is merely illustrative, and a heat buffer sheath 11 is located around the culture vessel 10. a If the system has the functionality to set the number of vertical grooves parallel to the central axis of the hollow cylinder, it may be one, two, three, or five or more. Furthermore, the gap V gap The retainer used as a gap setting means for setting the gap is the inner cylinder 11 illustrated in Figure 9(a). d1 The grooves may be transverse grooves perpendicular to the central axis of the hollow cylinder, and the number of transverse grooves provided on the outer circumference of the holder can be arbitrarily set. Furthermore, as a structure of the holder used in the second modification of the first embodiment, the inner cylinder 11 illustrated in Figure 9(c) f1 Even with a structure having spiral grooves on the outer circumference of a hollow cylinder, the heat buffer sheath 11 is located around the culture vessel 10. a Setting gap V gap This can function as a gap setting method to improve temperature stability by setting a specific value.

[0064] As shown in Figure 11, the left end of the storage cavity of the outer skin portion (21, 23, 23b, 25a, 25b) constituting the second modified example of the first embodiment has a bottom, forming a closed container with a stopper recess. A housing lid 23 is detachably provided on the open right end of the outer skin housing 21, forming a lidded container. Although not shown in the illustration, the structure is the same as that of the internal insertion type production tool 1a according to the first embodiment, with two flexible wing portions 25a and 25b attached to the left end of the outer skin portion (21, 23) to function as stoppers for insertion and retention inside the body of a cow 9, similar to the structure shown in Figure 1A.

[0065] According to the internally insertable production device 1b of the first embodiment, the orientation of the culture container 10 is aligned with the longitudinal central axis of the storage cavity, and the gap V is increased to a volume V = V1 + V2 + ΔV. gap This can be set up in a symmetrical structure around the culture vessel 10. Therefore, the heat buffer sheath 11 made of a gas layer a The volume V around the culture test tube 10a, including the area around the holder 24b, increased, creating a gap V. gap It can be set with a symmetrical structure. For this reason, according to the internally inserted production device 1b of the second modified example of the first embodiment, the bovine body temperature T b While ensuring good heat flow from, the temperature T of the culture fluid 8 e Rapid changes are further suppressed, and the good temperature stability of the culture vessel 10 can be further improved. That is, according to the internally inserted production device 1b according to the second modification of the first embodiment, the thermal time constant τ th This further increases the transient response changes due to heat flow, resulting in a remarkable effect of further enhancing temperature stability, as demonstrated by the bovine body temperature T b This can also be achieved by improving the efficiency of heat flow.

[0066] Note that although not shown in Figure 11, the cow vagina 9 in Reference Example 2 of Table 1 v Considering the possibility of the internally inserted production device detaching from the inside, it is preferable to attach a plurality of stoppers, such as umbrella-shaped ones, that have a shape that generates mechanical resistance in the direction of detachment to the outer skin of the internally inserted production device 1b according to the second modified example of the first embodiment, in order to prevent detachment. v The inner diameter may vary depending on the growth stage of the cow 9 and whether or not it has given birth. For this reason, like Russian Matryoshka dolls, multiple outer skin parts with different outer diameters are prepared so that the outer skin can be made into a multi-layered nesting structure, and the largest outer skin part is set to the cow vagina 9 v The inner diameter may be adjustable. If multiple outer skin parts with different outer diameters are provided, the inner outer skin part may have a structure that omits the wing parts 25a and 25b. Instead of a multi-layered nesting structure like a Matryoshka doll, multiple cylindrical diameter-increasing spacer tubes with different outer diameters are provided for the inner outer skin part, and the cow vagina 9 vThe effective outer diameter of the outer shell can be adjusted in relation to the inner diameter of the inner shell by using a multilayer structure. In the case of a Matryoshka doll or a multilayer structure using a diameter-increasing spacer cylinder, adding a stopper to the outermost layer of the outer shell to prevent detachment can more effectively prevent the internally inserted production device from falling out.

[0067] Note that Figure 11 shows an example in which the culture container 10 is held near the center by a single holder 24b and the direction and position of the long axis of the culture container 10 are adjusted, but this is merely an example. For example, as shown in Figure 1F, a first insertable production device 1c according to the first modification of the first embodiment may be arranged at both ends of the culture container 10 in the longitudinal direction, with a first holder 24b1 and a second holder 24b2 each having grooves. That is, the first holder 24b1 and the second holder 24b2 each having grooves are arranged opposite each other along the main axis in the longitudinal direction of the storage cavity, and the culture container 10 is held in a hollow state, and the heat buffer sheath 11 a As an internally implantable production device 1c, the thermal transient response can be slowed and temperature stability can be improved. Although not shown in the figure, in a structure held by two first holders 24b1 and second holders 24b2, a thermal buffer sheath 11 made of a gas layer a However, the heat buffer sheath 11 has a symmetrical structure in which the thickness is uniform in the radial direction perpendicular to the longitudinal direction of the culture vessel 10, including the areas of the first holder 24b1 and the second holder 24b2. a You can set it.

[0068] (Internal implantable production device according to the second embodiment) In the second embodiment of the present invention, an implantable production device has an electronic circuit board placed inside a storage cavity surrounded by an outer shell, together with a culture vessel 10. The electronic circuit board may be a monolithic semiconductor chip or a hybrid printed circuit board, but it is preferable that it has at least one of an information transmission function or an information acquisition / storage function. For example, if the electronic circuit board is equipped with an information transmission function as a transmitter, it becomes a hybrid medical device that enables simultaneous detection of rupture of membranes and in vitro fertilization egg production, as described in Patent Document 2. Even in the case of non-pregnant cows, if the electronic circuit board is a transmitter, it becomes a hybrid medical device that enables simultaneous detection of abnormalities such as shedding and in vitro fertilization egg production based on signals from the transmitter. Furthermore, if the electronic circuit board is equipped with an information acquisition function and storage function as a temperature sensor with data storage function, it can be used as a monitor to investigate the cause in the event of failure of in vitro fertilization egg production. When the electronic circuit board is a temperature sensor with data storage function, it is preferable to place the temperature sensor in close contact with the culture vessel 10 so that the thermal contact resistance between the temperature sensor and the culture vessel 10 is small.

[0069] It goes without saying that the electronic circuit board may have both an information transmission function and an information acquisition / storage function. According to the second embodiment of the implantable production device, the implantable production device, in which the electronic circuit board is housed together with the culture vessel 10, is inserted into the cow vagina 9 v By implanting the device inside the body of a cow 9, the process of producing in vitro fertilized eggs from cows 9 at low cost and the process of collecting associated monitoring information such as shedding and changes in temperature environment can be carried out in parallel. In the in-vivo production device according to the second embodiment, the culture container 10 is placed inside the storage cavity of the outer skin portion (21, 23) shown in Figure 1B via a holder 24a, and a thermal buffer sheath 11 made of a gas such as air or Ar gas surrounds the culture container 10. a This configuration enhances temperature stability, and an electronic circuit board is further embedded in the remaining space inside the storage cavity. Alternatively, the culture container 10 is positioned inside the storage cavity of the outer shell (21,23) shown in Figure 1F, with both ends held by the first holder 24a1 and the second holder 24a2, and a thermal buffer sheath 11 made of gas is placed around the culture container 10. aThis configuration enhances temperature stability, and the remaining space inside the storage cavity is used to house an electronic circuit board.

[0070] For example, if an electronic circuit board is used as a radio wave signal transmitter, the internally inserted production device according to the second embodiment is inserted into the cow vagina 9 v By inserting the device into the body of the cow 9 via the device, a configuration similar to the water rupture detection system already proposed by the present inventors as shown in Figures 1A and 1B of Patent Document 2 can be realized. Furthermore, by having a relay device for radio waves positioned at a distance of a first distance from the cow 9 and a receiving device positioned at a different distance of a second distance from the cow 9, a detection system equivalent to the water rupture detection system described in Patent Document 2, which detects abnormalities such as the detachment of the internally inserted production device, can be realized. As explained in Patent Document 2, by positioning the relay device at a first distance of 30m, preferably 10m, from the cow 9, and positioning the receiving device at a relatively long distance, an abnormality detection system can be constructed. The relay device receives a first radio wave signal of a first frequency and first power from a transmitter built into the outer casing 21 of the internally inserted production device together with the culture vessel 10, and the relay device converts the first radio wave signal into a second radio wave signal of a second frequency and a second power greater than the first power, and the second radio wave signal can be received by the receiving device. For example, the relay device may be placed inside the barn at a distance of a first distance from the cows 9, the receiving device may be placed in a control room away from the barn, and the second distance defined between the receiving device and the relay device may be longer than the first distance.

[0071] If cow 9 is pregnant, until calving begins, cow vagina 9 vThe process of producing in vitro fertilized eggs from cows 9 inexpensively can be carried out using an internally implantable production device placed inside the cow's body. The first radio wave signal from a transmitter built into the internally implantable production device inserted into the vagina is blocked from propagation by absorption and attenuation by the cow's body and does not reach the relay device that receives the first radio wave signal. However, just before the start of calving, i.e., when the amniotic sac ruptures or when calving has started, part or all of the transmitter is pushed out of the vagina and exposed, and the first radio wave signal is transmitted outside the vagina and reaches the relay device. The first radio wave signal received by the relay device is converted into a second radio wave signal, and when the second radio wave signal is received by the receiving device, information such as the start of calving is notified to the farmer assisting with calving. Patent Document 2 exemplifies a first frequency that is included in the 2.4 GHz band and a second frequency that is included in the 920 MHz band. The relay device and receiving device may be further configured to include a wireless LAN adapter.

[0072] According to the internally implantable production device of the second embodiment, the bovine body temperature T b By effectively utilizing the heat flow and providing a thermal buffer sheath, the thermal time constant in the heat flow is increased, resulting in good temperature stability of the culture vessel. In addition to this effect, the hybrid design can also incorporate additional functions. Specifically, it enables monitoring of issues such as the detachment of the internally inserted production device and changes in the temperature environment during embryo production, and allows for in vitro embryo production from cattle 9 without the use of expensive carbon dioxide culture equipment. Therefore, according to the internally inserted production device of the second embodiment, it enables producers to perform more reliable and dependable work, reduces producer stress and increases income, and facilitates the spread of inexpensive embryo transfer technology and a transformation of the in vitro embryo production process.

[0073] (Other embodiments) As described above, the present invention has been described by first and second embodiments, etc., but the descriptions and drawings that constitute part of this disclosure should not be understood as limiting the present invention. Various alternative embodiments, examples, and operational techniques will become apparent to those skilled in the art from this disclosure. Furthermore, it is possible to combine the technical ideas described in the first and second embodiments, etc., already mentioned. For example, the technical idea of ​​adjusting the effective outer diameter of an implantable production device using a multilayer structure such as a Matryoshka doll or a multilayer structure using a diameter-increasing spacer cylinder, as described in the second modification of the first embodiment, is also applicable to the implantable production device according to the first embodiment, the first modification of the first embodiment, and the second embodiment.

[0074] Thus, the present invention naturally includes various embodiments and the like that are not described herein. Therefore, the technical scope of the present invention is determined solely by the inventive features relating to the claims that are appropriate from the above description. [Explanation of symbols]

[0075] 1a, 1b, 1c...Intra-arterial production device, 8...Culture fluid (mixture of culture medium and culture gas phase), 9...Cow, 9 v ...cow vagina, 10...culture container, 10a...culture test tube, 10b...test tube cap, 11 a …Thermal buffer sheath 11 b1 ,11 c1 ,11 d1 ,11 e1 ,11 f1 ...Insertion tube, 11 b2 ,11 c2 ,11 f2 ...Suscepta, 11 x1 ...spacer, 11 x2 ...Spacer cap, 12...Operating string, 21...Outer casing, 23...Casing lid, 23a...String connection part, 241, 242, 243, 244...Vertical grooves, 24a, 24b...Holders, 24a1, 24b1...First holder, 24a2, 24b2...Second holder, 25a...First wing part, 25b...Second wing part

Claims

1. An outer shell comprising a substantially cylindrical outer shell housing having a recessed end, and a housing lid provided on the open end side of the outer shell housing to create a closed space over the recess, A culture vessel is housed inside the storage cavity formed by the aforementioned closed space, A gap setting means is provided inside the storage cavity, and within the storage cavity, it defines the position and orientation of the culture vessel, thereby creating a gap around the culture vessel. An internally inserted production device comprising: a culture container containing fertilized or unfertilized eggs together with a culture fluid; the outer casing containing the culture container being placed inside the body of a cow via the cow's vagina; and the fertilized or unfertilized eggs being cultured using the cow's body temperature.

2. The implantable production device according to claim 1, characterized in that an electronic circuit board is housed inside the storage cavity together with the culture vessel.

3. The steps include: placing fertilized eggs in a culture test tube with a culture medium, covering the culture test tube with a test tube cap, and preparing a culture vessel to be used in the developmental culture process; The process involves using a gas phase injector to replace the gas inside the culture vessel used in the developmental culture process so that the composition of the culture gas phase is similar to that of the bovine body, thereby forming and filling the culture fluid inside the culture vessel. The process involves: storing the culture container inside the storage cavity of the outer shell via a gap setting means so that it is in a predetermined orientation and position; setting a gap around the culture container using the gap setting means; and embedding a heat buffer sheath made of gas in the gap, thereby constituting an internally inserted production device for the generation and cultivation process; The process involves placing the internally inserted production device inside the body of a cow through the cow's vagina and using the cow's body temperature to carry out the embryo development and culture process. A method for preparing fertilized egg for transfer, characterized by including the following.

4. The steps include: placing the collected unfertilized eggs together with the culture medium into a culture test tube for maturation culture, covering the culture test tube with a test tube cap, and preparing the culture vessel for maturation culture to be used in the maturation culture process; The steps include: using the gas phase injector to replace the gas inside the culture vessel used in the maturation culture process, thereby forming and filling the culture fluid inside the culture vessel with a mature culture fluid; A step of setting a gap around the culture vessel for maturation culture using a gap setting means for maturation culture, embedding a heat buffer sheath made of gas in the gap, and constructing an internally insertable production device for maturation culture for the maturation culture process, The process involves placing the internally inserted production device for maturation culture inside the body of the cow, using the cow's body temperature to perform the maturation culture process of the unfertilized eggs, and producing mature eggs that can undergo the fertilization process. The embryo transfer preparation method according to claim 3, further comprising the steps including for the production of the embryo.

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