JP7495680B2 - Method for producing offspring from mammalian implanted fertilized eggs and pseudopregnancy induction device used therein - Google Patents

Description

The present invention relates to a method for producing offspring from transplanted fertilized eggs of mammals and a pseudopregnancy induction device used in this method.

The most commonly used experimental animals in the field of life science research are rodents such as rats, mice, and hamsters. Among them, rats and mice have long been used in a variety of biological experiments. For example, mice have been used in genetics and immunology research due to the existence of many inbred strains and congenic strains that have developed from them. Furthermore, in the field of developmental engineering, which has seen dramatic technological innovation in recent years, the ease of producing transgenic mice has led to the creation of a variety of human disease model animals, making them indispensable in medical research.

On the other hand, rats are about 10 to 15 times larger than mice in terms of body weight, and therefore have been used in biochemical experiments that require large amounts of material, such as purifying enzymes derived from organs, and in pathological research because of the ease of dissection. In addition, because rats exhibit physiological characteristics similar to those of humans, they are used in medical and drug discovery research as models of human diseases such as hypertension and diabetes, and in recent years many disease model strains have been created using genome editing technology.

These rodents have high reproductive capacity among mammals. One of the reasons for this is their short sexual cycle. The sexual cycle (menstrual cycle) is the interval between ovulations, and in humans it lasts from 24 to 32 days. Mammals have complete and incomplete sexual cycles; the former consists of a series of processes including the follicular phase, ovulation, and luteal phase, while the latter is a short sexual cycle that lacks the luteal phase.

In humans and other animals with complete sexual cycles, ovulation is preceded by the follicular phase, during which estrogen secretion increases. Ovulation is followed by the luteal phase, during which progesterone secretion increases. Progesterone is a type of luteal hormone that is necessary to induce implantation proliferation in the female endometrium and to maintain pregnancy.

On the other hand, animals with incomplete sexual cycles include rodents such as rats, mice, and hamsters mentioned above, as well as dotodactyls such as rabbits and pikas, insectivores such as the house shrew, and carnivores such as cats. In these animals, the corpus luteum formed after ovulation functionally regresses without the ability to secrete progesterone (luteal hormone), resulting in a sexual cycle that lacks a luteal phase as mentioned above, and the maturation of the follicles begins immediately in preparation for the next ovulation. Therefore, for example, rats and mice have a sexual cycle consisting of four phases - proestrus, estrus, metestrus, and diestrus - repeated in a short period of just about four days.

In the course of research into the sexual cycles of animals with incomplete sexual cycles, it became clear that in rats, mice, and other animals, mating results in the secretion of progesterone, and that the secreted progesterone suppresses ovulation for a certain period of time, thereby maintaining pregnancy. In other words, in some animals with incomplete sexual cycles, mating itself is an essential element in creating the physiological environment necessary to maintain pregnancy.

In recent years, the field of developmental engineering has been actively engaged in the creation of transgenic animals by introducing specific genes. In particular, mice are easy to create transgenic individuals, and a variety of model animals have been produced. On the other hand, rats are delicate, so it is difficult to create transgenic individuals even by applying the methods used for mice. In addition to traditional mating, transgenic rat individuals are systematically produced by genetically modifying fertilized eggs created by in vitro fertilization or intracytoplasmic sperm injection, and then implanting the resulting fertilized eggs into the oviduct and uterus of recipient female rats to introduce new genetic traits. In the case of embryo transplantation in rats, as mentioned above, if the female recipient is not stimulated to mate, progesterone will not be secreted and pregnancy cannot be maintained. For this reason, transgenic rats were previously produced by mating a male rat with a vas ligated (spermless) with a recipient female rat to stimulate mating and promote the secretion of progesterone, and then implanting the fertilized eggs.

However, in the above conventional method, it is necessary to prepare male rats that have had their vas ligated in advance just to provide mating stimulation to the females, which is undesirable from the perspective of reducing the number of experimental animals, and it also requires a lot of expense and labor, and cannot be said to be good in terms of work efficiency or cost-effectiveness. In addition, the female rat into which the fertilized eggs are to be implanted and the vas ligated male rat must be kept in the same cage, and mating behavior cannot be artificially forced, so it is left to the male rat. Therefore, it depends on the compatibility between the male and female rats and the success rate of mating behavior, so there is a need to reduce the time and improve the reliability.

On the other hand, after mating in rats or mice, the formation of a copulatory plug is confirmed, and an increase in the amount of progesterone and the associated suppression of ovulation are observed, but the animal does not actually become pregnant. This phenomenon is known as "pseudopregnancy," and research into its mechanism has been ongoing. Various methods for inducing pseudopregnancy have been proposed (for example, see Non-Patent Documents 1-3).

Non-Patent Document 1 proposes a method in which a glass rod is inserted and withdrawn into the vagina of a female rat, and the cervix is physically stimulated by hand.

Non-Patent Document 2 proposes a method of inducing pseudopregnancy by inserting a bipolar copper wire electrode into the vagina of a rat and passing a specified alternating current through it three times for five seconds. With this method, it is said that pseudopregnancy can be induced 100% of the time by stimulating with a voltage of 16 V or more.

Non-Patent Document 3 claims that pseudopregnancy can be induced by applying vibrations with a frequency of about 120 cycles per second to the genitals of female rats as a pseudo-copulatory stimulus.

However, in the method described in Non-Patent Document 1, the glass rod is moved manually, so the rate at which pseudopregnancy is induced varies depending on the skill of the practitioner, and there are problems with reproducibility and reliability. For this reason, there is a demand for the development of a device or technique that can more reliably induce a pseudopregnancy state.

The method described in Non-Patent Document 2 can easily and almost certainly induce pseudopregnancy, but since the technical feature is that a bipolar copper electrode is inserted into the rat's vagina and an electric current is passed through it, it is considered desirable to replace it with an alternative technique from the perspective of animal welfare for laboratory animals in recent years.

The method described in Non-Patent Document 3 leaves room for improvement in terms of the frequency of the vibration stimulation and the method of application.

In addition, the methods described in Non-Patent Documents 1 to 3 all aim to study the biological phenomenon of pseudopregnancy itself, and their industrial applications have not necessarily been fully explored.

De Feo, V. J.: Endocrinology 72:303(1963) Shichiro Sugawara, Saburo Takeuchi: Bulletin of the Japanese Society of Animal Science 35: 80 (1964) De Feo, V. J.: Endocrinology 79:440(1966)

In light of the above, the inventors have pursued intensive research and have developed an instrument equipped with a vibration generator that can be inserted into the vagina of a female rat and vibrated to the cervix to provide a simulated mating stimulus, thereby enabling the artificial induction of pseudopregnancy, and a method of inducing pseudopregnancy using the instrument. Furthermore, the inventors have found that when fertilized eggs at a specific developmental stage are transplanted into pseudopregnant female rats obtained using the instrument and the method of inducing pseudopregnancy using the instrument, normal offspring are obtained at a rate comparable to that of conventional methods. Furthermore, the inventors have found that pseudopregnancy can be induced in female rats even on the day of the transplantation of the fertilized eggs, and that normal offspring are obtained by the transplantation of the fertilized eggs.

The present invention was made based on the above findings, and aims to provide a simple, reliable and versatile method for inducing artificial pseudopregnancy in mammals and a method for producing offspring from transplanted fertilized eggs into pseudopregnant animals, which can be applied not only to laboratory animals such as rats but also to other animal species such as livestock. Another aim of the present invention is to provide an artificial pseudopregnancy induction device for producing offspring from transplanted fertilized eggs in mammals that is small, lightweight and easy to operate.

According to the present invention, in order to solve the above problems, the following technical methods or means are provided.
[1] The method for producing offspring from a transplanted fertilized egg of a mammal of the present invention is characterized by comprising at least the following steps <a> and <b>:

<a> a step of inserting a pseudopregnancy induction device for a mammal into the vagina of a mammal in the proestrus or estrus stage, bringing a tip of the pseudopregnancy induction device into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce a pseudopregnancy;
<b> after the step <a>, a step of implanting a fertilized egg at any developmental stage from the pronuclear stage to the morula stage into the oviduct and uterus of the mammal.
[2] In the method of the present invention for producing offspring from an implanted fertilized egg of a mammal, it is considered that the mammal is preferably an animal having an incomplete cycle.
[3] In addition, in the method of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is preferable that the imperfect cycle animal is a species selected from the group consisting of Rodentia, Decapoda, and Insectivora.
[4] Furthermore, in the method of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is preferable to transplant a two-cell stage fertilized egg in the step <b> within 12 to 24 hours after completion of the step <a>.
[5] In addition, in the method of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is preferable to transplant a pronuclear stage fertilized egg in the step <b> within 20 to 24 hours after the completion of the step <a>.
[6] Furthermore, in the method of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is considered preferable that the vibration stimulation in the step (a) is 18,000 to 25,000 times per minute.
[7] In the method for producing offspring from a transplanted fertilized egg of a mammal of the present invention, it is preferable that the vibration stimulation in the step <a> is a stimulation of 30 seconds repeated 2 to 7 times, with an interval of 30 seconds to 5 minutes between each stimulation.
[8] Furthermore, in the method for producing offspring from a transplanted fertilized egg of a mammal of the present invention, it is considered preferable that the vibration stimulus in the step <a> is given once for 30 to 120 seconds.
[9] The pseudopregnancy induction device for producing offspring from a transplanted fertilized egg of a mammal according to the present invention comprises:
A vibration generating device including a motor with an eccentric weight;
a main body having a motor housing portion that houses the motor;
an insertion member that is detachably attached to the tip end of the main body and can be inserted into the vagina of a mammal;
The present invention is characterized by comprising:
[10] In the pseudopregnancy induction device of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is considered that the mammal is preferably an animal having an incomplete cycle.
[11] In addition, in the pseudopregnancy induction device of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is considered that the animal with an incomplete sexual cycle is preferably one species selected from the group consisting of Rodentia, Decapoda, and Insectivora.
[12] Furthermore, in the pseudopregnancy induction device of the present invention for producing offspring from a transplanted fertilized egg of a mammal, it is considered preferable that the vibration generated by the motor is 18,000 to 25,000 times per minute.
[13] In the pseudopregnancy induction device of the present invention for producing offspring from the implanted fertilized egg of a mammal, it is considered preferable that the insertion member is made of an elastic material.
[14] Furthermore, in the pseudopregnancy induction device for producing offspring from the implanted fertilized egg of a mammal of the present invention, it is considered preferable that the tip of the elastic body is provided with a metallic contact end portion.

The present invention provides a simple, reliable, and versatile method for inducing artificial pseudopregnancy in mammals and a method for producing offspring from transplanted fertilized eggs into pseudopregnant animals, which can be applied not only to laboratory animals such as rats but also to other animal species such as livestock. The present invention also provides an artificial pseudopregnancy induction device for producing offspring from transplanted fertilized eggs in mammals that is small, lightweight, and easy to operate.

FIG. 1 is a schematic perspective view of a pseudopregnancy induction device for producing offspring from a transplanted fertilized egg of a mammal, which is one embodiment of the present invention. FIG. 2 is an exploded perspective view of FIG. 1 . FIG. 2 is a schematic cross-sectional view of FIG. 1. 1A and 1B are schematic perspective views showing an insertion member of a pseudopregnancy induction device for producing offspring from transplanted fertilized eggs of a mammal.

The present invention will be described in detail below with reference to the embodiments.

In this specification, the term "artificial pseudopregnancy induction" means inducing a pseudopregnancy in a female mammal by using a pseudopregnancy induction device to provide a stimulation equivalent to the stimulation of mating to a female mammal, without actually mating the male and female mammals.
In addition, in the present specification, the term "proestrus" refers to the initial stage in the next sexual cycle when the corpus luteum of the diestrus phase disappears. During this stage, the development of follicular follicles begins rapidly and protrudes onto the surface of the ovary.
Furthermore, in this specification, the term "estrus" refers to the period during which mammals mate and during which preparations are made for ovulation of eggs from the follicles developed during the proestrus period. For example, in the case of mice, changes in appearance, such as redness and swelling around the external genitalia, appear from the proestrus period to the estrus period.

The method for producing offspring from a transplanted fertilized egg of a mammal of the present invention is characterized by including at least the following steps <a> and <b>. That is, it includes the steps of: <a> inserting a mammalian pseudopregnancy induction device into the vagina of a mammal in the proestrus or estrus stage, bringing the tip of the pseudopregnancy induction device into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce a pseudopregnancy; <b> after step <a>, transplanting a fertilized egg at any developmental stage from the pronuclear stage to the morula stage into the oviduct and uterus of the mammal.

Step <a> is a step of applying vibration stimulation to the cervix of a mammal using a mammal pseudopregnancy induction device. Incompletely cycled animals are preferably considered to be mammals to which the method of producing offspring from transplanted fertilized eggs of a mammal of the present invention can be applied. Incompletely cycled animals are preferably, for example, rodents such as rats, mice, and hamsters, as well as dotodactyls such as rabbits and pikas, insectivores such as suncus (shrew), and carnivores such as cats, and among these, rats and mice, which are used in large quantities as experimental animals, are particularly preferred.

To confirm whether such mammals are in proestrus or estrus, any conventional method can be used. For example, in the case of rats or mice, the experimenter can place his or her hand on the back of a female rat or mouse or rhythmically vibrate the base of the tail to confirm whether the animal exhibits a lordosis reflex, which causes the animal to arch its back in a large arch, or by ear wiggling. The lordosis reflex is observed not only in rodents such as rats and mice, but also in carnivorous animals such as cats. For mammals in which the lordosis reflex does not occur, a method of confirming whether the animal is in proestrus or estrus can be exemplified by measuring the concentration of hormones such as estrogen.

After confirming that the mammal is in proestrus or estrus using the above method, a pseudopregnancy induction device is inserted into the mammal's vagina, the tip of the pseudopregnancy induction device is gently placed against the mammal's cervix, and vibration stimulation is applied to the cervix. When applying the vibration stimulation, a stopwatch or the like is used to check the stimulation presentation time. It is also preferable to slide the pseudopregnancy induction device back and forth in small movements to further stimulate the cervix.

There are no particular limitations on the external shape or power supply method of the pseudopregnancy induction device, so long as it can provide vibration stimulation to the cervix of the mammal, but since it is necessary to operate the device while holding the mammal, it is desirable for the device to be small, lightweight, and cordless. In addition, since the tip of the pseudopregnancy induction device is inserted into the vagina of the mammal, it is desirable for the device to have water resistance and waterproofing so that it can be washed with water after use. Furthermore, it is preferable that only the tip is removable, and that the tip is made of a material that is resistant to chemicals such as rubbing alcohol, or a material that can be sterilized by boiling or autoclaving. It is also preferable that the tip is disposable, so that it can be discarded after one use.

As such a pseudopregnancy induction device, for example, it is preferable to consider a configuration including a vibration generating device consisting of a motor with an eccentric weight, as described below, a main body having a motor housing that houses the motor, and an insertion member that can be inserted into the vagina of a mammal and is detachably attached to the tip of the main body.

The vibration stimulation in step <a> is preferably, for example, 18,000 to 25,000 times per minute. If the vibration stimulation is within the above range, pseudopregnancy can be reliably induced by short-term stimulation presentation, but it is not necessarily limited to the above numerical range.

As a preferred example of the vibration stimulation in step <a>, a stimulation of 30 seconds is repeated 2 to 7 times, with an interval of 30 seconds to 5 minutes between each stimulation. It is preferable to disinfect the pseudopregnancy induction device after each use.

Taking the mating behavior of rats as an example, male rats will repeatedly insert and ejaculate multiple times over the course of one night. This cycle of multiple insertions and ejaculations is called an "ejaculation series." The inventors have investigated the number of insertions in an ejaculation series and have confirmed that if a male rat inserts less than seven times, pregnancy does not occur and the rat returns to its sexual cycle, but if insertions are confirmed to occur seven or more times, pregnancy occurs almost 100% of the time.

On the other hand, the inventors have confirmed that in experiments to induce artificial pseudopregnancy in mammals using a pseudopregnancy induction device described below, it is not essential to present vibration stimuli seven or more times. Specifically, they have confirmed that pseudopregnancy can be induced almost 100% by applying vibration stimuli for 30 seconds, followed by a 30 second interval, followed by a 30 second stimulus (total of 90 seconds). They have also confirmed that pseudopregnancy can be induced more reliably by applying vibration stimuli for 30 seconds, followed by a 30 second interval, followed by a 30 second stimulus, followed by a 30 second interval, followed by a 30 second stimulus (total of 150 seconds).

The interval between stimulations is set based on the refractory period after one ejaculation in the ejaculation series until the next insertion becomes possible.

In addition, as the vibration stimulation in step <a>, for example, a single stimulation of 30 to 120 seconds without an interval is also a preferred example. When artificially inducing pseudopregnancy in a mammal using the pseudopregnancy induction device of the present invention, pseudopregnancy can be induced in a shorter time and more efficiently than stimulation during natural mating. Specifically, a single stimulation of 30 seconds to the cervix can induce pseudopregnancy with a success rate of approximately 60%. Furthermore, the longer the stimulation presentation time, the higher the success rate of pseudopregnancy induction, reaching nearly 100% at 120 seconds.

As mentioned above, the high efficiency of this pseudopregnancy induction is due to the fact that the tip of the pseudopregnancy induction device is brought into contact with the cervix of the mammal and vibration stimulation is applied directly to the cervix.

As described above, the method for artificially inducing pseudopregnancy in mammals of the present invention can induce pseudopregnancy almost reliably, and therefore it is no longer necessary to prepare a male mammal with a vas ligation operation, which was previously required only to provide mating stimulation to a female mammal, and it is possible to reduce the number of experimental animals. In addition, it is possible to reduce the cost and labor required for vas ligation and the like, and it is possible to significantly improve work efficiency and cost-effectiveness. Furthermore, it is no longer necessary to insert an electrode wire into the vagina of a mammal and determine the presence or absence of a vaginal smear by measuring electrical conductivity, which was previously performed to confirm pregnancy and pseudopregnancy. Therefore, the method for artificially inducing pseudopregnancy in mammals of the present invention is excellent from the viewpoint of animal protection, and moreover, the number of steps required for producing pseudopregnant animals is reduced compared to the conventional method, making it possible to improve work efficiency.

After step <a>, in step <b>, a fertilized egg at any stage of development from the pronuclear stage to the morula stage is implanted into the oviduct and uterus of a mammal.

The fertilized egg may be a naturally fertilized egg resulting from mating between a male and female mammal, or may be a fertilized egg artificially fertilized by in vitro fertilization or intracytoplasmic sperm injection. In particular, when genetic manipulation or the like is involved, it is preferable to use a naturally fertilized egg.

The eggs used to create fertilized eggs by in vitro fertilization or ICSI may be fresh eggs collected from the ovaries of female mammals, or may be cryopreserved eggs. Furthermore, the sperm used to create fertilized eggs by ICSI may be fresh sperm collected from male mammals, or may be cryopreserved sperm.

For the transplantation of fertilized eggs, fertilized and developed eggs using the above methods may be immediately used for transplantation, or frozen fertilized eggs may be used.

Various conventional methods can be used to collect fertilized eggs, such as a method of flushing the inside of the oviduct with a liquid medium used for culturing fertilized eggs.

As for the method of collecting semen, various methods known in the art can be applied, such as the massage method, electrical stimulation method, water pressure method, and artificial vagina method.

The developmental stage of the fertilized egg is not particularly limited, so long as the rate at which normal offspring can be obtained from the transplanted fertilized egg by the method of the present invention is comparable to that of the conventional method, i.e., approximately 40 to 60%. For example, the developmental stage is preferably any of the pronuclear stage to morula stage, particularly any of the pronuclear stage to two-cell stage. In particular, according to the method of the present invention, even when using a pronuclear fertilized egg, which had a relatively low rate of obtaining normal offspring in the conventional method of producing offspring by fertilized egg transplantation, the rate at which normal offspring can be obtained from the transplanted fertilized egg exceeds about 40%.
Furthermore, even when using morulae, which have traditionally yielded a relatively low rate of normal offspring, it is possible to obtain normal offspring from transplanted fertilized eggs at a level comparable to that obtained by conventional methods.

In the method of producing offspring from a transplanted fertilized egg by inducing artificial pseudopregnancy in a mammal of the present invention, it is preferable to transplant a two-cell stage fertilized egg as the step <b> within 12 to 24 hours, preferably 17 to 20 hours, after the completion of the step <a>. The interval between the steps <a> and <b> is almost synchronized with the timing at which a naturally fertilized fertilized egg develops to the two-cell stage as a result of mating between a male and a female mammal. Therefore, the oviduct and uterus of a mammal provide an optimal physiological environment for a two-cell stage transplanted fertilized egg, making it easier to obtain normal offspring. For example, a time schedule can be exemplified in which the practitioner (experimenter) performs the treatment of step <a> on a mammal in proestrus around the evening on the day before the planned date of transplantation of the fertilized egg, and the practitioner (experimenter) performs the treatment of step <b> from morning to evening on the day of the planned date of transplantation.

In addition, in the method of producing offspring from a transplanted fertilized egg by inducing artificial pseudopregnancy in a mammal of the present invention, it is preferable to transplant a pronuclear fertilized egg as the step <b> within 20 to 24 hours after the completion of the step <a>. The interval between the steps <a> and <b> is slightly different from the timing at which a naturally fertilized fertilized egg develops to the pronuclear stage as a result of mating between a male and a female mammal. Specifically, the oviduct and uterus of a mammal are in a physiological environment suitable for a fertilized egg that is at a more advanced stage of development than the pronuclear stage. However, the oviduct and uterus of a mammal adjust to an optimal physiological environment according to the developmental stage of the transplanted fertilized egg, and a synchronization of timing occurs so that the physiological environment in the oviduct and uterus does not change any further to a physiological environment suitable for a fertilized egg at a later developmental stage until the developmental stage of the transplanted fertilized egg progresses. In other words, it can be said that the mother's side adjusts the physiological environment to that of the transplanted fertilized egg. Therefore, even when a pronuclear fertilized egg is transplanted, which has traditionally been less effective at producing normal offspring than a two-cell fertilized egg, it is now easier to produce normal offspring, and in fact a higher percentage of normal offspring can be obtained than when a two-cell fertilized egg is transplanted. For example, an example of a time schedule is one in which the practitioner (experimenter) performs the treatment of step <a> on a mammal in proestrus around the evening of the day before the planned date for transplantation of the fertilized egg, and the practitioner (experimenter) performs the treatment of step <b> in the evening of the planned date for transplantation.

On the other hand, in the method of the present invention for producing offspring from a transplanted fertilized egg by inducing artificial pseudopregnancy in a mammal, if the mammal is in the estrus phase of its sexual cycle, on the day of the procedure for transplanting a fertilized egg into the mammal, even if the interval between step <a> and step <b> is within 1 to 6 hours, preferably within 1 to 2 hours, a vibration stimulus can be applied to the cervix of the mammal to induce an artificial pseudopregnancy suitable for transplantation of the fertilized egg. When inducing an artificial pseudopregnancy on the day of embryo transplantation, examples of the developmental stage of the fertilized egg suitable for transplantation include the two-cell stage or the pronuclear stage.

In this way, the method of the present invention for producing offspring from transplanted fertilized eggs by inducing artificial pseudopregnancy in mammals is not restricted by the time course of the fertilized egg's developmental stages after fertilization and implantation, which was essential to consider in conventionally known methods, and offers greater freedom than conventional methods, making it possible to reduce the burden on the practitioner.

The following is a detailed explanation of a mammalian pseudopregnancy induction device optimized for use in such a method for producing offspring from transplanted fertilized eggs by artificially inducing pseudopregnancy in mammals, with reference to the drawings.

Figure 1 is a schematic perspective view of a pseudopregnancy induction device for producing offspring from a mammalian implanted fertilized egg, which is one embodiment of the present invention. Figure 2 is an exploded perspective view of Figure 1. Figure 3 is a schematic cross-sectional view of Figure 1.

The pseudopregnancy induction device 1 for producing offspring from transplanted fertilized eggs of mammals (hereinafter simply referred to as the pseudopregnancy induction device 1) comprises a vibration generating device 2 consisting of a motor with an eccentric weight, a main body 4 having a motor housing 3 for housing the motor, and an insertion member 5 that is detachably attached to the tip 41 of the main body 4 and can be inserted into the vagina of a mammal.

The main body 4 has a vibration generator 2 for applying vibrations to the insertion member 5 and a battery storage section 46, and is provided with an exterior case 42 that is attached to the main body 4 as shown in Figures 1 to 3.

The tip 41 of the main body 4 is tapered to form the insert member mounting cylinder 43. The insert member mounting cylinder 43 is a cylindrical cylinder with a lid, and communicates with the cylindrical space S2 (see FIG. 2) of the insert member 5. As shown in FIG. 3, the insert member mounting cylinder 43 is formed on the top surface of the main body 4 so that its central axis is biased toward the rear surface 4a of the main body 4 relative to the central axis of the main body 4.

A through hole 44 is formed through the front surface 4b of the main body 4, and a space S1 is formed directly below the through hole 44, and the through hole 44 and the space S1 are blocked by a flexible switch member 45. The switch member 45 is made of an elastomer resin and is fused to the opening edge of the through hole 44. Therefore, the switch member 45 blocking the through hole 44 can be pushed into the space S1 from the outside with a finger.

A battery accommodating section 46 is formed extending from the end of the main body 4 opposite the insertion member mounting shaft tube 43. The battery accommodating section 46 has an anode terminal metal fitting 46a at its bottom and a cathode terminal spring 46b extending from the body 47 at its top. When a battery B is accommodated in the battery accommodating section 46, the battery B is accommodated and held within the battery accommodating section 46 in a state where it is electrically connected to the anode terminal metal fitting 46a and the cathode terminal spring 46b. The battery accommodating section 46 is protected by the main body case 42, which is a detachable, bottomed cylinder.

Battery B may be a commercially available dry cell, or a secondary battery such as a removable lithium ion battery. In the case of a dry cell, it is preferable for it to be a relatively small and lightweight battery such as an AA or AAA battery.

A board fixing portion 48 is formed and extends from the upper side of the body portion 47. A printed wiring board 49 is fixed to the board fixing portion 48. A switch SW that is electrically connected to the anode terminal metal fitting 46a and the cathode terminal spring 46b is mounted on the printed wiring board 49. The switch SW mounted on the printed wiring board 49 is mounted in a position facing the switch member 45 provided on the exterior case 42. Therefore, when the switch member 45 is pressed with a finger, the switch SW is turned on and off by the pressed switch member 45.

The motor housing 3 is formed to extend above the board fixing portion 48. When the motor housing 3 is installed in the space S1 of the insert member mounting shaft tube 43, the outer peripheral surface of the motor housing 3 is in close contact with the inner peripheral surface of the insert member mounting shaft tube 43.

The motor M is accommodated in the motor accommodating section 3. When the motor accommodating section 3 is installed inside the space S1 of the insert member mounting shaft tube 43, the outer peripheral surface of the portion of the motor M that is open from the motor accommodating section 3 abuts against the inner peripheral surface of the insert member mounting shaft tube 43 via the spacer 31. The rotating shaft of the motor M protrudes into the space S1 from the top surface of the motor accommodating section 3, and an eccentric weight 32 is fixed to the protruding rotating shaft. The motor M equipped with such an eccentric weight 32 is used as the vibration generating device 2.

When the switch SW mounted on the printed wiring board 49 is turned on, the voltage of the battery B is applied to the motor M, causing it to rotate. When the motor M rotates, the eccentric weight 32 fixed to the rotating shaft rotates eccentrically around the rotating shaft, and this eccentric rotation causes the rotating shaft to vibrate, causing the motor M itself, which is housed and held in the motor housing 3, to vibrate. This vibration of the motor M is then transmitted via the motor housing 3 to the insert member mounting shaft tube 43, which is in close contact with the motor housing 3.

The vibrations generated by the motor M are not particularly limited as long as they are capable of inducing pseudopregnancy, but for example, a vibration frequency of 18,000 to 25,000 times per minute is preferably considered. If the vibration stimulation is within the above range, pseudopregnancy can be reliably induced by presenting the stimulation for a short period of time, but it is not necessarily limited to the above numerical range.

The exterior case 42 is made of a plastic material such as ABS resin or PP resin, and has a generally circular cross section in the short direction so that it is easy for the operator to grip. The battery storage section 46 of the main body 4 is attached to the internal space through the upper opening. The inner peripheral surface shape of the upper opening of the exterior case 42 is the same shape as the lower end of the body 47, so that the battery storage section 46 and the lower end of the body 47 are fitted inside the exterior case 42.

When the battery storage section 46 and the lower end of the body section 47 are fitted inside the outer case 42, the O-ring O of the annular groove provided at the lower end of the body section 47 elastically deforms into the upper opening of the outer case 42, providing watertight and elastic support between the battery storage section 46 and the body section 47 and the outer case 42. By attaching the main body section 4 to the outer case 42, the gripping section of the pseudopregnancy induction device 1 is formed by the outer case 42, and the battery B held in the battery storage section 46 is hermetically stored by the outer case 42.

The insert member 5 is attached to the upper end portion 41 (insertion member mounting shaft tube 43) of the main body 4, which has the outer case 42 formed in this manner.

The insertion member 5 attached to the tip 41 (insertion member mounting shaft tube 43) of the main body 4 has a fitting hole 52 formed in the lower end surface 51a of the base end 51. The insertion member mounting shaft tube 43 formed in the tip 41 of the main body 4 is press-fitted into the fitting hole 52 so as to be freely attached and detached. When the insertion member mounting shaft tube 43 is fitted, the outer peripheral surface 43a of the insertion member mounting shaft tube 43 is tightly attached to the inner peripheral surface 52a of the fitting hole 52. Therefore, the vibration of the motor M transmitted to the insertion member mounting shaft tube 43 via the motor housing 3 is transmitted from the base end 51 of the insertion member 5 through the body 54 to the tip 53 of the insertion member 5 that abuts against the cervix of the mammal.

As shown in FIG. 4(a), the body 54 of the insertion member 5 is formed so that the diameter is constant from the tip 53 to the base end 51 of the insertion member 5, and the diameter becomes larger near the base end 51, forming a tapered surface. The tip of the insertion member 5 is rounded, so that it will not damage organs when inserted into the vagina of a mammal or when it abuts against the cervix. The diameter and length of the insertion member 5 are not particularly limited as long as they do not damage organs when inserted into the vagina of a mammal or when it abuts against the cervix, and the size can be changed appropriately depending on the target animal species. For example, an insertion member for rats has a diameter near the tip of about 5 mm and a length of the part inserted into the rat's vagina of about 7 cm.

The material of the insertion member 5 is not particularly limited, but it is preferably an elastic body so as not to damage the organs when it is inserted into the vagina of a mammal or when it comes into contact with the cervix. Examples of elastic bodies include silicone resin and synthetic rubber. In particular, it is preferable that the insertion member 5 is made of silicone resin, which has excellent chemical resistance and heat resistance, considering sterilization treatment using disinfectants such as rubbing alcohol, and sterilization and sterilization treatment such as boiling or autoclaving. Furthermore, it is also preferable that the insertion member 5 is disposable so that it can be discarded after one use.

As shown in FIG. 4(b), it is also preferable to provide a metallic contact end 5a at the tip 53 of the insertion member 5. The shape of the contact end 5a is not particularly limited as long as it does not damage the oviduct and vagina of the mammal, but it is preferable that it is spherical. As shown in FIG. 4(b), in order to prevent the contact end 5a from falling off from the insertion member 5 inside the mammal, for example, the contact end 5a may be exposed in a semi-spherical shape from the tip of the insertion member 5, with the remaining part embedded in the insertion member 5. The material of the contact end 5a is not particularly limited as long as it does not have a negative effect on the mammal that is the mother, but for example, it may be made of a metal with excellent corrosion resistance, such as stainless steel or titanium with high biocompatibility.

Mammals to which the pseudopregnancy induction device of the present invention can be applied are incompletely sexually active animals. For example, rodents such as rats, mice, and hamsters, as well as dotodactyls such as rabbits and pikas, insectivores such as the suncus (shrew), and carnivores such as cats are preferred, and among these, rats and mice, which are used in large numbers as experimental animals, are particularly preferred.

The pseudopregnancy induction device of the present invention, having the above-mentioned configuration, can be applied not only to laboratory animals but also to other animal species such as livestock, and is small, lightweight, and easy to operate. Furthermore, the pseudopregnancy induction device of the present invention is not limited in any way by the above-mentioned embodiment in terms of its external shape, power supply method, etc., as long as it can provide vibration stimulation to the cervix of a mammal. Furthermore, since the pseudopregnancy induction process in the present invention requires that the mammal be held in place while the operation is performed, it is desirable that the pseudopregnancy induction device be small, lightweight, and cordless in structure, eliminating the need for a power cord.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples in any way.
Example A: Comparison of the method for producing offspring from implanted fertilized eggs of a mammal in which pseudopregnancy has been induced by the method of the present invention with the conventional method
(1) Test animals For the collection of fertilized eggs and the subsequent embryo transfer, rats of the Iar:Wister-Imamichi strain (Animal Reproduction Research Institute, a foundation) were used. All animals were kept in plastic cages in a room with constant temperature and humidity (temperature 23°C ± 3°C, humidity 50% ± 10%) and light and darkness control (lights on from 7:00 to 19:00). All animal care and experimental procedures were carried out in accordance with the Iwate University Animal Experiment Guidelines and with the approval of the Iwate University Animal Research Committee.

(2) Collection of fertilized eggs Female rats aged 8 to 13 weeks were randomly selected and, to induce superovulation, human chorionic gonadotropin (hCG, Asuka Animal Health Co., Ltd.) was intraperitoneally injected at a concentration of 300 IU/kg, 48 hours after which pregnant mare serum gonadotropin (PMSG, Asuka Animal Health Co., Ltd.) was intraperitoneally injected at a concentration of 300 IU/kg. The female rats were then mated overnight with mature male rats aged 10 weeks or older. If a vaginal plug was present in the female rat, it was determined that mating had definitely taken place.

Twenty-four hours after the injection of hCG, pronuclear rat fertilized eggs were collected by flushing the oviduct with PB1 medium. Multiple rat fertilized eggs were cultured in fresh HTF medium at 37° C. in an environment of 5% CO ₂ and 95% air until they reached the two-cell stage.

(3) Creation of female rats in which pseudopregnancy was artificially induced Female rats in the proestrus stage, 8 to 13 weeks of age, were used to artificially induce pseudopregnancy. The pseudopregnancy induction device of the present invention was inserted into the vagina of the female rat, and the insertion member of the pseudopregnancy induction device was gently pressed against the cervix of the uterus to apply vibration stimulation. Stimulation with the pseudopregnancy induction device was performed for 30 seconds, a total of 7 times, with a 5-minute interval between each stimulation. This interval time was based on the refractory period after ejaculation in male rats. As a control group, female rats in which artificial pseudopregnancy had been induced by mating with vasoligated male rats were used.

(4) Fertilized Egg Transplantation In Example 1, fertilized eggs that had developed to the two-cell stage were transplanted into the oviducts of pseudopregnant female rats 12 hours after artificial pseudopregnancy was induced using a pseudopregnancy induction device.

In addition, in Example 2, 12 hours after artificial pseudopregnancy was induced using a pseudopregnancy induction device, two-cell stage fertilized eggs that had been frozen and stored were transplanted into the oviducts of pseudopregnant female rats.

Furthermore, in Example 3, 24 hours after artificial pseudopregnancy was induced using a pseudopregnancy induction device, pronuclear fertilized eggs were transplanted into the oviducts of pseudopregnancy-induced female rats.

As control group 1, fertilized eggs that had developed to the two-cell stage were transferred into the oviducts of pseudopregnant female rats 12 hours after mating with vasoligated male rats.
As control group 2, 12 hours after mating with a vasoligated male rat, frozen two-cell stage fertilized eggs were transferred into the oviducts of pseudopregnant female rats.

(5) Data Analysis All data were subjected to chi-square test followed by Ryan's multiple comparison test.

For the pseudopregnant female rats of Examples 1 to 3 and Controls 1 and 2, the implantation rate of the transplanted fertilized eggs and the number of normally developed offspring were counted on the 21st day of pregnancy. The results are shown in Table 1.

As shown in Table 1, in Example 1, 63% of the transplanted fresh fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 54% of the fertilized eggs. In Example 2, 46% of the transplanted frozen fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 24% of the fertilized eggs. In Example 3, 73% of the transplanted fresh fertilized eggs at the pronuclear stage implanted, and normal offspring were obtained from 67% of the fertilized eggs.

In female rats that were mated with vasoligated male rats in control group 1 to artificially induce pseudopregnancy, 72% of the transplanted fresh fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 63% of the fertilized eggs.

In addition, in female rats in which artificial pseudopregnancy was induced by mating with vasoligated male rats in control group 2, 56% of the transplanted frozen fertilized eggs implanted, and normal offspring were obtained from 32% of the fertilized eggs. Comparing these results with those of control groups 1 and 2, it was confirmed that the female rats in which artificial pseudopregnancy was induced in the present embodiment gave birth to normal offspring at a rate similar to that obtained by the conventional method.

Example B: Comparison of implantation rate and normal birth rate by stimulation method
In Example 4, fresh two-cell stage fertilized eggs were implanted into the oviducts of pseudopregnant female rats in the same manner as in Example 1, except that the vibration stimulation pattern to the cervix was presented once for 120 seconds.

In Example 5, fresh two-cell stage fertilized eggs were implanted into the oviducts of pseudopregnant female rats in the same manner as in Example 1, except that the vibration stimulation was presented to the cervix twice in total for 30 seconds, with a 30-second interval between each stimulation.

In Example 6, fresh pronuclear fertilized eggs were implanted into the oviducts of pseudopregnant female rats in the same manner as in Example 3, except that the vibration stimulation to the cervix was presented twice for 30 seconds each, with a 30-second interval between each stimulation.

In Example 7, the vibration stimulation was applied to the cervix twice for 30 seconds, with a 30-second interval between each stimulation. 96 hours after artificial pseudopregnancy induction using a pseudopregnancy induction device, fresh morulae were transferred to the oviducts of pseudopregnancy-induced female rats in the same manner as in Example 1.

For the pseudopregnant female rats of Examples 4 to 7, the implantation rate of the transplanted fertilized eggs and the number of normally developed offspring were counted on the 21st day of pregnancy. The results are shown in Table 2.

As shown in Table 2, in Example 4, 64% of the transplanted fresh fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 40% of the fertilized eggs. In Example 5, 65% of the transplanted fresh fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 62% of the fertilized eggs. In Example 6, 52% of the transplanted fresh fertilized eggs at the pronuclear stage implanted, and normal offspring were obtained from 43% of the fertilized eggs. In Example 7, 30% of the transplanted fresh morulae implanted, and normal offspring were obtained from 21% of the fertilized eggs.

Example C: Comparison of implantation rate and normal birth rate by cervical stimulation on the day of fertilized egg (embryo) transfer
In Example 8, fresh two-cell stage fertilized eggs were transplanted into the oviducts of pseudopregnant female rats in the same manner as in Example 5, except that the stimulation was presented on the day of fertilized egg (embryo) transplantation, specifically 1 to 2 hours prior to the transplantation.

In addition, in Example 9, fresh fertilized eggs in the pronuclear stage were transplanted into the oviducts of pseudopregnant female rats in the same manner as in Example 6, except that the stimulation was presented on the day of fertilized egg (embryo) transfer, specifically 5 to 6 hours prior.

For the pseudopregnant female rats of Examples 8 and 9, the implantation rate of the transplanted fertilized eggs and the number of normally developed offspring were counted on the 21st day of pregnancy. The results are shown in Table 3.

As shown in Table 3, in Example 8, 75% of the transplanted fresh fertilized eggs at the two-cell stage implanted, and normal offspring were obtained from 68% of the fertilized eggs. In Example 9, 72% of the transplanted fresh fertilized eggs at the pronuclear stage implanted, and normal offspring were obtained from 67% of the fertilized eggs.

Therefore, it was confirmed that the method of the present invention for inducing artificial pseudopregnancy in mammals and the method for producing offspring from fertilized eggs transplanted into pseudopregnant animals produce normal offspring with a success rate equal to or higher than that of conventional methods. In particular, it was confirmed that in mammals in which vibration stimulation was applied to the cervix on the day of fertilized egg (embryo) transplantation, the implantation rate of fertilized eggs exceeded 70%, and normal offspring were produced from 65% or more of the fertilized eggs. Furthermore, it was confirmed that even when pronuclear stage fertilized eggs and morulae, which had a low success rate in the past, were transplanted, normal offspring were produced at a rate almost comparable to that of conventional methods.

1 Pseudopregnancy induction device 2 Vibration generator 3 Motor housing 31 Spacer 32 Eccentric weight 4 Main body 4a Rear surface 4b Front surface 41 Tip 42 Outer case 43 Insert member mounting shaft tube 43a Outer circumferential surface 44 Through hole 45 Switch member 46 Battery housing 46a Anode terminal fitting 46b Cathode terminal spring 47 Body 48 Board fixing portion 49 Printed wiring board 5, 5' Insert member 5a Contact end 51 Base end 51a Lower end surface 52 Fitting hole 52a Inner circumferential surface 53 Tip 54 Body B Battery M Motor O O-ring S1, S2 Space SW Switch

Claims (6)

At least the following steps <a><b> :
<a> a step of inserting a pseudopregnancy induction device for a mammal into the vagina of a mammal in the proestrus or estrus stage, bringing a tip of the pseudopregnancy induction device into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce a pseudopregnancy;
<b> after the step <a>, a step of implanting a fertilized egg at any stage of development from the pronuclear stage to the morula into the oviduct and uterus of the mammal ,
The vibration stimulation in the step <a> is 18,000 to 25,000 times per minute;
The vibration stimulation in the step <a> is a stimulation for 30 seconds repeated 2 to 7 times, with an interval of 30 seconds to 5 minutes between each stimulation.
A method for producing offspring from a transplanted fertilized egg of a mammal, comprising the steps of: At least the following steps <a><b> :
<a> a step of inserting a pseudopregnancy induction device for a mammal into the vagina of a mammal in the proestrus or estrus stage, bringing a tip of the pseudopregnancy induction device into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce a pseudopregnancy;
<b> after the step <a>, a step of implanting a fertilized egg at any stage of development from the pronuclear stage to the morula into the oviduct and uterus of the mammal ,
The vibration stimulation in the step <a> is 18,000 to 25,000 times per minute;
As the vibration stimulus in the step <a>, a stimulus of 30 to 120 seconds is applied once.
A method for producing offspring from a transplanted fertilized egg of a mammal, comprising the steps of: 3. The method for producing offspring from an implanted fertilized egg of a mammal according to claim 1 or 2 , wherein the mammal is an animal having an incomplete cycle. 4. The method for producing offspring from a transplanted fertilized egg of a mammal according to claim 3 , wherein the animal with an imperfect cycle is one species selected from the group consisting of Rodentia, Decapoda, and Insectivora. 3. The method for producing offspring from a mammalian transplanted fertilized egg according to claim 1 or 2, characterized in that, in the step <b>, a two-cell stage fertilized egg is transplanted within 12 to 24 hours after the completion of the step <a>. 3. The method for producing offspring from a mammalian transplanted fertilized egg according to claim 1 or 2 , characterized in that, in step <b>, a pronuclear stage fertilized egg is transplanted within 20 to 24 hours after completion of step <a>.

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