Pseudopregnancy inducer for producing offspring from transplanted fertilized eggs of mammals

Abstract

To provide a small-sized lightweight and easily-operable artificial false pregnancy induction device for childbirth production from a transplanted fertilized egg of a mammal.SOLUTION: A false pregnancy induction device for childbirth production from a transplanted fertilized egg of a mammal includes a vibration generator system comprising a motor with an eccentric weight, a body part having a motor storage part for storing the motor, and an insertion member attachable to / detachable from a tip part of the body part, and insertable into the vagina of a mammal. Vibration generated by the motor is 18,000-25,000 times per minute.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing offspring from a fertilized egg implanted in a mammal and a pseudopregnancy induction device used therein. [Background technology]

[0002] 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 genetic and immunological research due to the existence of many inbred strains and congenic strains that have developed from them. In addition, in the field of developmental engineering, where technological innovation has progressed dramatically in recent years, the ease of producing transgenic mice has led to the creation of various human disease model animals, making them indispensable in medical research.

[0003] 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 the purification of enzymes derived from organs, and in pathological and disease research because of the ease of dissection. In addition, because rats exhibit physiological characteristics similar to 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.

[0004] These rodents have a 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. Mammalian sexual cycles are classified into complete and incomplete sexual cycles, the former consisting 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.

[0005] In complete cyclic animals including humans, the follicular phase precedes ovulation, and estrogen secretion increases. After ovulation, it is called the luteal phase, and progesterone secretion increases. Progesterone is a type of luteal hormone necessary to induce implantation proliferation in the female endometrium and maintain pregnancy.

[0006] On the other hand, incomplete cyclic animals include rodents such as rats, mice, and hamsters mentioned above, as well as lagomorphs such as rabbits and pikas, insectivores such as shrews (soricids), and carnivores such as cats. In these animals, the corpus luteum formed after ovulation regresses functionally without having the ability to secrete progesterone (luteal hormone). Therefore, as described above, the sexual cycle lacks the luteal phase, and follicle maturation starts immediately for the next ovulation. For this reason, for example, in rats and mice, the sexual cycle consisting of four stages: proestrus, estrus, metestrus, and diestrus, is repeated in a short period of only about four days.

[0007] In the process of research on the sexual cycle of such incomplete cyclic animals, in rats, mice, etc., it has been clarified that as a result of copulation, luteal hormone is secreted, and the secreted luteal hormone suppresses ovulation for a certain period, thus maintaining pregnancy. That is, in some incomplete cyclic animals, copulation itself is an essential factor in preparing the physiological environment for maintaining pregnancy.

[0008] In recent years, in the field of developmental engineering, the creation of transgenic animals into which specific genes have been introduced has been actively carried out. Among them, in the case of mice, since it is easy to create transgenic individuals, a variety of model animals have been produced. On the other hand, since rats are delicate, it is difficult to create transgenic individuals even if the methods used for mice are applied. Transgenic rats are created by genetically modifying fertilized eggs produced by in vitro fertilization or intracytoplasmic sperm injection methods in addition to classical mating, and then transplanting the obtained fertilized eggs into the fallopian tubes and uterus of recipient female rats to introduce new genetic traits in a planned manner. In the transplantation of fertilized eggs in rats, as described above, if the recipient female is not given a mating stimulus, luteinizing hormone is not secreted and pregnancy cannot be maintained. Therefore, conventionally, transgenic rats have been created by mating a male rat with its vas deferens ligated (without sperm) with a recipient female rat in advance to give a mating stimulus to promote the secretion of luteinizing hormone, and then transplanting the fertilized eggs.

[0009] However, in the above conventional method, it is necessary to prepare a male rat with a vas deferens ligation surgery in advance only to give a mating stimulus to the female, which is not preferable from the viewpoint of reducing experimental animals, and moreover, it requires a lot of cost and labor, and the working efficiency and cost-effectiveness are not good. In addition, it is necessary to breed a female rat for transplanting fertilized eggs and a vasectomized male one by one in the same breeding cage, and moreover, the mating behavior cannot be artificially forced and is left to the male rat. Therefore, it has been influenced by the compatibility between male and female rats and the success rate of mating behavior, and shortening of time and improvement of certainty have been demanded.

[0010] On the other hand, after mating rats, mice, etc., although the formation of a copulatory plug is confirmed, and an increase in the amount of luteinizing hormone and accompanying suppression of ovulation are observed, there is a known phenomenon called "pseudopregnancy" in which pregnancy does not actually occur, and research on its mechanism has been underway. So far, various methods for inducing pseudopregnancy have been proposed (see, for example, Non-Patent Documents 1-3).

[0011] In Non-Patent Document 1, a method has been proposed in which a glass rod is inserted into and withdrawn from the vagina of a female rat, and the uterine cervix is physically stimulated manually.

[0012] In Non-Patent Document 2, a method has been proposed in which a bipolar electrode of a copper wire is inserted into the vagina of a rat, and a predetermined alternating current is applied three times for 5 seconds to induce pseudopregnancy. In this method, it is said that 100% pseudopregnancy can be induced by stimulating with a voltage of 16 V or higher.

[0013] In Non-Patent Document 3, it is said that pseudopregnancy can be induced by applying vibrations at a frequency of about 120 cycles / second to the genital organs of a female rat as a pseudo-copulation stimulus.

[0014] However, in the method described in Non-Patent Document 1, since the glass rod is moved manually, the induction rate of pseudopregnancy varies due to differences in the skills of the operator, etc., and there are difficulties in reproducibility and certainty. Therefore, there has been a demand for the development of an apparatus and a technique that can more reliably induce a pseudopregnant state.

[0015] In the method described in Non-Patent Document 2, although pseudopregnancy can be induced simply and almost certainly, since it is characterized by inserting a bipolar electrode of a copper wire into the vagina of a rat and applying an electric current, it is considered desirable to replace it with an alternative technique from the viewpoint of animal welfare for experimental animals in recent years.

[0016] In the method described in Non-Patent Document 3, there was room for improvement in the vibration frequency (frequency) and application method of the vibration stimulus.

[0017] In addition, the methods described in Non-Patent Documents 1 to 3 are all aimed at studying the life phenomenon of pseudopregnancy itself, and their industrial applications have not necessarily been fully studied.

Prior Art Documents

Non-Patent Documents

[0018]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0019] Against the above background, as a result of intensive research, the inventors of the present invention have found that by inserting an instrument equipped with a vibration generator into the vagina of a female rat and applying vibration to the cervix, a pseudo-copulation stimulus can be given, and an instrument capable of artificially reproducing pseudo-pregnancy induction and a method for inducing pseudo-pregnancy using the same have been developed. Furthermore, when the inventors transplanted fertilized eggs at a specific developmental stage into female rats induced to be pseudo-pregnant by the above-described instrument and the method for inducing pseudo-pregnancy using the same, they found that normal offspring could be obtained at a rate comparable to the conventional method. And also, the inventors have found that even on the day of transplantation of fertilized eggs, pseudo-pregnancy can be induced in female rats, and moreover, normal offspring can be obtained by transplantation of fertilized eggs.

[0020] The present invention has been made based on the above findings, and an object thereof is to provide a method for inducing artificial pseudo-pregnancy in mammals and a method for producing offspring from transplanted fertilized eggs into pseudo-pregnant animals, which are simple, reliable, and highly versatile, and can be applied not only to experimental animals such as rats but also to other animal species such as livestock. Another object of the present invention is to provide an artificial pseudo-pregnancy inducer for producing offspring from transplanted fertilized eggs in mammals, which is small, lightweight, and easy to operate.

Means for Solving the Problems

[0021] According to the present invention, in order to solve the above problems, the following technical methods or technical means are provided. 〔1〕The method for producing offspring from a transplanted fertilized egg of a mammal according to the present invention includes at least the following steps Characterized by including.

[0022] A step of inserting a mammalian pseudopregnancy inducer into the vagina of a mammalian in the proestrus to estrus stage, bringing the tip of the pseudopregnancy inducer into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce pseudopregnancy; The above step After that, a step of transplanting 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 for producing offspring from a transplanted fertilized egg of a mammal of the present invention, it is preferably considered that the mammal is an anestrous cyclic animal. 〔3〕Further, in the method for producing offspring from a transplanted fertilized egg of a mammal of the present invention, it is preferably considered that the anestrous cyclic animal is one selected from the group consisting of Rodentia, Lagomorpha, and Insectivora. 〔4〕Furthermore, in the method for producing offspring from a transplanted fertilized egg of a mammal of the present invention, the step After completion, within 12 to 24 hours, the said process As such, it is preferably considered to transplant a fertilized egg at the two-cell stage. 〔5〕In addition, in the method for producing a progeny from a transplanted fertilized egg of a mammal of the present invention, the step Within 20 to 24 hours after the completion of the above process As such, it is preferably considered to transplant a fertilized egg in the pronuclear stage. 〔6〕Furthermore, in the method for producing offspring from a transplanted fertilized egg of a mammal according to the present invention, the step It is preferably considered that the vibration stimulation in [the relevant context] is 18,000 to 25,000 times per minute. 〔7〕Also, in the method for producing offspring from a transplanted fertilized egg of a mammal according to the present invention, the said step As the vibration stimulation in [the above situation], it is preferably considered to repeat the stimulation of 30 seconds once for 2 to 7 times, and to insert an interval of 30 seconds to 5 minutes between the stimulations. 〔8〕Furthermore, in the method for producing offspring from a transplanted fertilized egg of a mammal according to the present invention, the above step As the vibration stimulation, it is preferably considered to give a stimulation of 30 seconds to 120 seconds once. 〔9〕The apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention comprises a vibration generating device composed of a motor with an eccentric weight, a main body portion having a motor housing portion for housing the motor, and an insertion member that is detachably attached to the tip of the main body portion and can be inserted into the vagina of a mammal. It is characterized by comprising the above. 〔10〕In the apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention, it is preferably considered that the mammal is an anestrous cyclic animal. 〔11〕Further, in the apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention, it is preferably considered that the anestrous cyclic animal is one selected from the group consisting of rodents, lagomorphs, and insectivores. 〔12〕Furthermore, in the apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention, it is preferably considered that the vibration generated by the motor is 18,000 to 25,000 times per minute. 〔13〕Also, in the apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention, it is preferably considered that the insertion member is an elastic body. 〔14〕Furthermore, in the apparatus for inducing pseudopregnancy for producing offspring from transplanted fertilized eggs of mammals according to the present invention, it is preferably considered that a metal contact end portion is provided at the tip of the elastic body.

Advantages of the Invention

[0023] According to the present invention, a simple, reliable, and highly versatile method for inducing artificial pseudopregnancy in mammals and a method for producing offspring from transplanted fertilized eggs in pseudopregnant animals that can be applied not only to experimental animals such as rats but also to other animal species such as livestock are realized. Further, according to the present invention, an artificial pseudopregnancy inducing device for producing offspring from transplanted fertilized eggs of mammals that is small, lightweight, and easy to operate is realized.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0025] Hereinafter, the present invention will be described in detail with reference to embodiments.

[0026] In this specification, the term "artificial induction of pseudopregnancy" means to give a female mammal a stimulus equivalent to copulation stimulation using an agent for inducing pseudopregnancy without actually mating the male and female mammals, and induce pseudopregnancy. Also, in this specification, "proestrus" means the first stage when the corpus luteum in the anestrus period disappears and enters the next estrous cycle. At this time, the development of follicles rapidly begins and protrudes from the ovarian surface. Furthermore, in this specification, "estrus" means the period when a mammal mates, and means the period when ovulation of an egg is prepared from the follicles developed in the aforementioned proestrus. For example, in the case of a mouse, external changes such as redness and swelling around the external genitalia appear from proestrus to estrus.

[0027] The method for producing offspring from a transplanted fertilized egg of a mammal according to the present invention includes at least the following steps is characterized by including. That is A step of inserting a mammalian pseudopregnancy inducer into the vagina of a mammalian in the proestrus to estrus stage, bringing the tip of the pseudopregnancy inducer into contact with the cervix of the mammal, and applying a vibration stimulus to the cervix to induce pseudopregnancy; The above step After that, it includes each step of transplanting a fertilized egg in any developmental stage from the pronuclear stage to the morula into the oviduct and uterus of the mammal.

[0028] Step This is a step of applying vibration stimulation to the cervix of a mammal using a mammalian pseudopregnancy inducer. As a mammal to which the method for producing offspring from transplanted fertilized eggs of the present invention can be applied, it is preferably considered to be an animal with an incomplete estrous cycle. Examples of animals with an incomplete estrous cycle include, for example, rodents such as rats, mice, and hamsters, as well as lagomorphs such as rabbits and pikas, insectivores such as shrews (soricidae), carnivores such as cats, etc. Among them, rats and mice, which are widely used as experimental animals, are particularly preferred.

[0029] As a method for confirming that such a mammal is in the proestrus to estrus stage, conventionally known methods can be appropriately used. For example, in the case of rats and mice, the experimenter can place a hand on the back of the female rat or mouse, or apply rhythmic vibration to the base of the tail, and confirm the presence or absence of the lordosis reflex, in which the back is curved in a large arch, or ear wiggling. The lordosis reflex is observed not only in rodents such as rats and mice but also in carnivores such as cats. For mammals in which the lordosis reflex does not occur, for example, methods such as measuring the concentration of hormones such as estrogen to confirm that they are in the proestrus to estrus stage are exemplified.

[0030] After confirming that the mammal is in the proestrus to estrus stage by the above method, a pseudopregnancy inducer is inserted into the vagina of the mammal, and the tip of the pseudopregnancy inducer is gently brought into contact with the cervix of the mammal, and vibration stimulation is applied to the cervix. When applying vibration stimulation, it is performed while checking the stimulation presentation time using a stopwatch or the like. It is also preferable to slide the pseudopregnancy inducer finely back and forth to further stimulate the cervix.

[0031] The pseudopregnancy induction device is not particularly limited in its external shape or power supply method, as long as it can provide vibration stimulation to the cervix of a mammal, but since it is necessary to operate the device while restraining the mammal, it is desirable that the device is small, lightweight, and cordless without a power cord. Furthermore, since the tip of the pseudopregnancy induction device is inserted into the vagina of a mammal, it is desirable that the device has 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.

[0032] As such a pseudopregnancy induction device, for example, a vibration generator consisting of a motor with an eccentric weight, which will be described later, It is preferable that the configuration include a living device, a main body having a motor housing portion that houses the motor, and an insertion member that is detachable from the tip of the main body and can be inserted into the vagina of a mammal.

[0033] process As the vibration stimulation in [the relevant context], for example, it is preferably considered to be 18,000 to 25,000 times per minute. If the vibration stimulation is within the above range, false pregnancy can surely be induced by presenting the stimulation for a short time, but it is not necessarily limited to the above numerical range.

[0034] Also, the step As the vibration stimulation, for example, it is preferably exemplified that the stimulation of 30 seconds per time is repeated 2 to 7 times, and an interval of 30 seconds to 5 minutes is interposed between the stimulations. In addition, it is desirable to disinfect the pseudopregnancy inducer each time it is used.

[0035] Taking the copulatory behavior of rats as an example, male rats repeat insertion and ejaculation multiple times overnight. Such a cycle of multiple insertions and ejaculations is called an "ejaculation series". When the present inventors investigated the number of insertions in a series of ejaculation series, they confirmed that when the number of insertions of male rats was less than 7 times, pregnancy did not occur and the estrous cycle returned, but when 7 or more insertions were confirmed, pregnancy was established almost 100%.

[0036] On the other hand, the present inventors have also confirmed that it is not always essential to present vibration stimulation 7 times or more in the artificial pseudopregnancy induction experiment of mammals using the pseudopregnancy inducer described below. Specifically, it has been confirmed that by applying vibration stimulation of 30-second stimulation, a 30-second interval, and 30-second stimulation (total 90 seconds), pseudopregnancy can be induced almost 100%. In addition, it has been confirmed that by applying vibration stimulation of 30-second stimulation, a 30-second interval, 30-second stimulation, a 30-second interval, and 30-second stimulation (total 150 seconds), pseudopregnancy can be induced more reliably.

[0037] In addition, the interval between the stimulations is set based on the refractory period time until the next insertion becomes possible after ejaculation once in the ejaculation series.

[0038] Also, the step As the vibration stimulation in [the above situation], for example, it is also preferably exemplified that a stimulation of 30 seconds to 120 seconds is given once without providing an interval. In the artificial induction of pseudopregnancy in mammals using the pseudopregnancy inducer of the present invention, pseudopregnancy can be induced in a shorter time and with higher efficiency than the stimulation in natural mating. Specifically, when a 30-second stimulation is given to the cervix once, pseudopregnancy can be induced with a success rate of approximately 60%. Also, the success rate of inducing pseudopregnancy increases as the stimulation presentation time becomes longer, reaching almost 100% at 120 seconds.

[0039] Such an increase in the efficiency of inducing pseudopregnancy is due to, as described above, bringing the tip of the pseudopregnancy inducer into contact with the cervix of a mammal and directly applying a vibration stimulation to the cervix.

[0040] As described above, in the method for artificially inducing pseudopregnancy in mammals of the present invention, since pseudopregnancy can be induced almost surely, male mammals that have undergone vasectomy, which were conventionally necessary only for giving mating stimulation to female mammals, become unnecessary, and the number of experimental animals can be reduced. Also, many costs and labor required for vasectomy and the like can be reduced, and the working efficiency and cost-effectiveness can be greatly improved. Furthermore, conventionally, for the purpose of confirming pregnancy and pseudopregnancy, judgments such as the presence or absence of a vaginal smear by inserting an electrode wire into the vagina of a mammal and measuring the electrical conductivity are unnecessary. Therefore, the method for artificially inducing pseudopregnancy in mammals of the present invention is excellent from the viewpoint of animal welfare, and moreover, in producing pseudopregnant animals, the number of steps is less than before, and the working efficiency can be improved.

[0041] Such a process After that, step As a method, a fertilized egg in any developmental stage from the pronuclear stage to the morula stage is transplanted into the fallopian tube and uterus of a mammal.

[0042] The fertilized egg may be a fertilized egg that has been naturally fertilized as a result of mating between a male and a female mammal, or may be a fertilized egg that has been artificially fertilized by an in vitro fertilization method or an intracytoplasmic sperm injection method. Among these, when genetic manipulation or the like is involved, it is preferable to use a fertilized egg that has been naturally fertilized.

[0043] The egg used for the production of a fertilized egg by an in vitro fertilization method or an intracytoplasmic sperm injection method may be a fresh egg collected from the ovary of a female mammal, or may be a cryopreserved egg. Also, the sperm used for the production of a fertilized egg by an intracytoplasmic sperm injection method may be fresh sperm collected from a male mammal, or may be cryopreserved sperm.

[0044] For the transplantation of a fertilized egg, the fertilized egg fertilized and developed by each of the above methods may be immediately used for transplantation, or a cryopreserved fertilized egg may be used.

[0045] Regarding the method for collecting a fertilized egg, various conventionally known methods can be applied. For example, a method of perfusing and flushing the fallopian tube using a liquid medium used for culturing the fertilized egg is exemplified.

[0046] Regarding the sperm collection method as well, various conventionally known methods can be applied. For example, a massage method, an electrostimulation method, a hydrostatic pressure method, an artificial vagina method, etc. are exemplified.

[0047] Regarding the developmental stage of the fertilized egg, as long as the proportion of obtaining normal offspring from the transplanted fertilized egg by the method of the present invention is at a level not inferior to the conventional method, that is, generally about 40 to 60%, there is no particular limitation. For example, it is preferably considered to be in any of the stages from the pronuclear stage to the morula stage, particularly in any of the developmental stages from the pronuclear stage to the two-cell stage. In particular, according to the method of the present invention, even when a fertilized egg in the pronuclear stage, for which the proportion of obtaining normal offspring was relatively low in the conventional method for producing offspring by fertilized egg transplantation, is used, the proportion of obtaining normal offspring from the transplanted fertilized egg exceeds about 40%. Furthermore, even when using morulae from which the proportion of normal parturitions was relatively low in the past, normal parturitions can be obtained from transferred fertilized eggs at a level comparable to that of conventional methods.

[0048] In the method for producing offspring from transferred fertilized eggs by artificially inducing pseudopregnancy in mammals according to the present invention, the above step After completion, within 12 to 24 hours, preferably within 17 to 20 hours, the said process is preferably considered to be the transplantation of a fertilized egg at the two-cell stage. The interval between such a step and stroke is almost synchronized with the timing when a fertilized egg naturally fertilized as a result of mating between a male and a female mammal develops to the two-cell stage. Therefore, the oviduct and uterus of a mammal are in an optimal physiological environment for the transplanted fertilized egg at the two-cell stage, and normal offspring are easily obtained. For example, around evening on the day before the scheduled date of fertilized egg transplantation, the operator (experimenter) performs step Perform the treatment, and from morning to evening on the day of the scheduled transplantation, the operator (experimenter) performs the process A time schedule for performing the treatment can be exemplified.

[0049] Further, in the method for producing offspring from a transplanted fertilized egg by inducing artificial pseudopregnancy in a mammal of the present invention, the step Within 20 to 24 hours from the completion of the above step is preferably considered to be the transplantation of a fertilized egg at the pronuclear stage to a mammal in the proestrus stage. The interval between such a step and stroke is slightly different from the timing when a fertilized egg naturally fertilized as a result of mating between a male and a female mammal develops to the pronuclear stage. Specifically, the oviduct and uterus of a mammal are in a physiological environment suitable for a fertilized egg at a more advanced developmental stage 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 timing synchronization occurs so that the physiological environment in the oviduct and uterus does not change to a physiological environment suitable for a fertilized egg at a later developmental stage until the developmental stage of the transplanted fertilized egg progresses. That is, it can be said that the maternal side adjusts the physiological environment to the transplanted fertilized egg. Therefore, conventionally, even in the transplantation of a fertilized egg at the pronuclear stage, which was more difficult to obtain normal offspring than the transplantation of a fertilized egg at the two-cell stage, normal offspring are easily obtained, and rather, normal offspring can be obtained at a higher rate than when a fertilized egg at the two-cell stage is transplanted. For example, around evening on the day before the scheduled date of fertilized egg transplantation, the operator (experimenter) performs step Treatment was performed, and the operator (experimenter) performed the procedure in the evening on the day scheduled for transplantation The time schedule for performing the treatment can be exemplified.

[0050] On the other hand, in the method for producing offspring from a transplanted fertilized egg by inducing artificial pseudopregnancy in a mammal according to the present invention, if the estrous cycle of the mammal has entered the estrus phase, on the day of the treatment of transplanting the fertilized egg into the mammal, for example, in step and stroke Even if the interval is within 1 to 6 hours, preferably within 1 to 2 hours, vibration stimulation can be applied to the cervix of a mammal to induce an artificial pseudopregnancy suitable for embryo transfer. When inducing an artificial pseudopregnancy on the day of embryo transfer, examples of the developmental stage of the embryo suitable for transfer are the two-cell stage or the pronuclear stage.

[0051] Thus, in the method for producing offspring from transferred embryos by inducing artificial pseudopregnancy in mammals of the present invention, it is not restricted by the time course of the developmental stage of the fertilized egg after fertilization and implantation, which was essential to consider in conventionally known methods. Compared with conventional techniques, it has a higher degree of freedom and can also reduce the burden on the operator.

[0052] The mammal pseudopregnancy inducer optimized for use in such a method for producing offspring from transferred embryos by inducing artificial pseudopregnancy in mammals will be described in detail below with reference to the drawings.

[0053] FIG. 1 is a schematic perspective view of a pseudopregnancy inducer for producing offspring from transferred embryos in a mammal according to an embodiment of the present invention. FIG. 2 is an exploded perspective view of FIG. 1. Further, FIG. 3 is a schematic cross-sectional view of FIG. 1.

[0054] The pseudopregnancy inducer 1 for producing offspring from transferred embryos in a mammal (hereinafter simply referred to as the pseudopregnancy inducer 1) includes a vibration generating device 2 composed of an eccentric weight motor, 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.

[0055] The main body 4 has a vibration generating device 2 for applying vibration to the insertion member 5 and a battery housing 46, and as shown in FIGS. 1 to 3, it includes an outer case 42 attached to the main body 4.

[0056] The tip 41 of the main body 4 has a reduced diameter to form an insertion member attachment shaft cylinder 43. The insertion member attachment shaft cylinder 43 is a covered cylindrical shaft cylinder and communicates with the cylindrical space S2 (see FIG. 2) of the insertion member 5. Further, as shown in FIG. 3, the insertion member attachment shaft cylinder 43 is formed on the upper surface of the main body 4 such that its central axis is offset toward the rear surface 4a side of the main body 4 with respect to the central axis of the main body 4.

[0057] 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. The through-hole 44 and the space S1 are closed 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 that closes the through-hole 44 from the outside with a finger can be pushed into the space S1.

[0058] A battery housing portion 46 is formed to extend at the end of the main body 4 opposite to the insertion member attachment shaft cylinder 43. The battery housing portion 46 is provided with an anode terminal fitting 46a at its bottom and a cathode terminal spring 46b extending from the body portion 47 at its upper part. When a battery B is housed in the battery housing portion 46, the battery B is housed and held in the battery housing portion 46 in a state of being electrically connected to the anode terminal fitting 46a and the cathode terminal spring 46b. The battery housing portion 46 is protected by a main body case 42 that is a detachable bottomed cylinder.

[0059] The battery B may be a commercially available dry battery or a secondary battery such as a detachable lithium-ion battery. In the case of a dry battery, it is preferably a relatively small and light one such as a AA battery or a AAA battery.

[0060] On the upper side of the body portion 47, a substrate fixing portion 48 is formed to extend. A printed wiring board 49 is fixed to the substrate fixing portion 48. A switch SW electrically connected to the anode terminal 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 at a position facing the switch member 45 provided on the exterior case 42. Therefore, when the switch member 45 is pushed with a finger, the switch SW is turned on and off by the pushed switch member 45.

[0061] On the upper side of the substrate fixing portion 48, a motor housing portion 3 is formed to extend. When the motor housing portion 3 is installed in the space S1 of the insertion member mounting shaft cylinder 43, the outer peripheral surface of the motor housing portion 3 is in close contact with the inner peripheral surface of the insertion member mounting shaft cylinder 43.

[0062] A motor M is housed in the motor housing portion 3. When the motor housing portion 3 is installed in the space S1 of the insertion member mounting shaft cylinder 43, the outer peripheral surface of the part of the motor M that is open from the motor housing portion 3 abuts against the inner peripheral surface of the insertion member mounting shaft cylinder 43 via the spacer 31. The rotating shaft of the motor M projects into the space S1 from the upper surface of the motor housing portion 3, and an eccentric weight 32 is fixed to the projected rotating shaft. The motor M provided with such an eccentric weight 32 is used as the vibration generating device 2.

[0063] When the switch SW mounted on the printed wiring board 49 is turned on, the voltage of the battery B is applied and the motor M rotates. When the motor M rotates, the eccentric weight 32 fixed to the rotating shaft rotates eccentrically about the rotating shaft as the center of rotation. Due to this eccentric rotation, the rotating shaft is shaken and the motor M itself housed and held in the motor housing portion 3 vibrates. Then, the vibration of this motor M is propagated to the insertion member mounting shaft cylinder 43 that is in close contact with the motor housing portion 3 via the motor housing portion 3.

[0064] The vibration generated by the motor M is not particularly limited as long as it can induce pseudopregnancy. For example, it is preferably considered to be 18,000 to 25,000 times per minute. If the vibration stimulation is within the above range, pseudopregnancy can be surely induced with a short-time stimulation presentation, but it is not necessarily limited to the above numerical range.

[0065] The exterior case 42 is a case made of a plastic material such as ABS resin or PP resin. The cross-section in the short side direction is substantially circular so that it is easy for an operator to grip. The battery housing portion 46 of the main body portion 4 is mounted in its internal space from the upper opening. The inner peripheral surface shape of the upper opening of the exterior case 42 is the same as the lower end portion of the body portion 47, and the lower end portions of the battery housing portion 46 and the body portion 47 are installed inside the exterior case 42.

[0066] Also, when the lower end portions of the battery housing portion 46 and the body portion 47 are installed inside the exterior case 42, the O-ring O in the annular groove provided at the lower end portion of the body portion 47 is elastically deformed to the upper opening of the exterior case 42 to provide watertight and elastic support between the battery housing portion 46, the body portion 47, and the exterior case 42. By attaching the main body portion 4 to the exterior case 42, the gripping portion of the pseudopregnancy inducer 1 is formed by the exterior case 42, and the battery B housed and held in the battery housing portion 46 is hermetically stored by the exterior case 42.

[0067] An insertion member 5 is attached to the upper tip portion 41 (insertion member attachment shaft cylinder 43) of the main body portion 4 provided with the exterior case 42 formed in this way.

[0068] The insertion member 5 attached to the tip 41 (insertion member attachment shaft cylinder 43) of the main body 4 has a fitting hole 52 formed in the lower end surface 51a of its base end 51. The fitting hole 52 is detachably press-fitted with the insertion member attachment shaft cylinder 43 formed at the tip 41 of the main body 4. The inner peripheral surface 52a of the fitting hole 52 is in close contact with the outer peripheral surface 43a of the insertion member attachment shaft cylinder 43 when the insertion member attachment shaft cylinder 43 is fitted. Therefore, the vibration of the motor M propagated to the insertion member attachment shaft cylinder 43 through the motor housing 3 is propagated from the base end 51 of the insertion member 5 through the body 54 to the tip 53 of the insertion member 5 that contacts the cervix of a mammal.

[0069] As shown in Fig. 4(a), the body 54 of the insertion member 5 is formed such that its diameter is constant from the tip 53 to the base end 51 of the insertion member 5, and the diameter becomes thicker near the base end 51 to form a tapered surface. And the tip of the insertion member 5 is rounded, and it will not damage the organs when inserted into the vagina of a mammal and when contacting the cervix. Regarding the diameter and length of the insertion member 5, there is no particular limitation as long as it does not damage the organs when inserted into the vagina of a mammal and when contacting the cervix, and the size can be appropriately changed according to the target animal species. For example, in the case of an insertion member for a rat, it is exemplified that the diameter near the tip is about 5 mm and the length of the portion inserted into the vagina of the rat is about 7 cm.

[0070] The material of the insertion member 5 is not particularly limited, but it is preferably considered to be an elastic body so as not to damage the organs when inserted into the vagina of a mammal and when contacting the cervix. Examples of the elastic body include silicon resin and synthetic rubber. Among them, considering sterilization treatment using disinfectants such as alcohol for disinfection, and sterilization and disinfection treatment such as boiling or autoclave treatment, it is particularly preferable that it is made of silicon resin with excellent chemical resistance and heat resistance. Furthermore, it is also preferable that the insertion member 5 is in a disposable mode that can be discarded every time it is used.

[0071] Also, as shown in Fig. 4(b), it is preferably considered that the tip 53 of the insertion member 5 is provided with a metal contact end portion 5a. The shape of the contact end portion 5a is not particularly limited as long as it does not damage the fallopian tubes and vagina of mammals, but a spherical shape is desirable. Further, as shown in Fig. 4(b), in order to prevent the contact end portion 5a from falling off the insertion member 5 inside the body of a mammal, for example, it is exemplified that the contact end portion 5a is exposed hemispherically from the tip of the insertion member 5 and the remaining portion is embedded in the insertion member 5. Further, the material of the contact end portion 5a is not particularly limited as long as it has no adverse effect on the mammalian mother and fetus, but for example, it is exemplified that it is made of a metal excellent in corrosion resistance such as stainless steel or titanium with high biocompatibility.

[0072] The mammals to which the false pregnancy inducer of the present invention can be applied are non-seasonal cyclic animals. For example, in addition to rodents such as rats, mice, and hamsters, it is preferably lagomorphs such as rabbits and Japanese white rabbits, insectivores such as shrews (Sorex araneus), carnivores such as cats, etc., and among them, rats and mice that are widely used as experimental animals are particularly preferred.

[0073] Due to having the configuration as described above, the false pregnancy inducer of the present invention can be applied not only to experimental animals but also to other animal species such as livestock, and moreover, it is small, lightweight, and easy to operate. Also as long as the false pregnancy inducer of the present invention can apply a vibration stimulus to the cervix of a mammal, the external shape, power supply method, etc. are not limited by the above-described embodiments at all. Furthermore, in the process of inducing false pregnancy in the present invention, since it is necessary to perform the operation while restraining the mammal, it is desirable that the false pregnancy inducer has a cordless structure that is small, lightweight, and eliminates the power cord.

Examples

[0074] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited by the following examples at all. <Example A: Comparison between the method for producing offspring from transplanted fertilized eggs of mammals induced to have false pregnancy by the method of the present invention and the conventional method> (1) Test animals For the collection of fertilized eggs and subsequent embryo transfer, Iar:Wistar-Imamichi strain rats {(Animal Breeding and Research Institute, Inc.)} were used. All animals were housed in a temperature- and humidity-controlled room (temperature: 23°C ± 3°C, humidity: 50% ± 10%) with a light-dark cycle (light on from 7:00 to 19:00) using plastic cages. In addition, the housing and experimental procedures for all animals were carried out in accordance with the guidelines of the Animal Experimentation of Iwate University and were approved by the Animal Research Committee of Iwate University.

[0075] (2) Collection of fertilized eggs Female rats aged 8 - 13 weeks were randomly selected. To induce superovulation, 48 hours after intraperitoneal injection of human chorionic gonadotropin (hCG, Asuka Animal Health Co., Ltd.) at a concentration of 300 IU / kg, pregnant mare serum gonadotropin (PMSG, Asuka Animal Health Co., Ltd.) was intraperitoneally injected at a concentration of 300 IU / kg. Subsequently, the female rats were 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 occurred successfully.

[0076] Twenty-four hours after the injection of hCG, pronuclear-stage rat fertilized eggs were collected by perfusing and flushing the oviducts using PB1 medium. Multiple rat fertilized eggs were cultured in fresh HTF medium until the two-cell stage under an environment of 37°C, 5% CO2, and 95% air.

[0077] (3) Creation of artificially induced pseudopregnant female rats Female rats in the proestrus stage aged 8 - 13 weeks were used to induce artificial pseudopregnancy. The pseudopregnancy inducer of the present invention was inserted into the vagina of the female rat, and the insertion member of the pseudopregnancy inducer was gently pressed against the cervix to apply vibration stimulation. The stimulation by the pseudopregnancy inducer was performed a total of 7 times for 30 seconds each, with a 5-minute interval between stimulations. The interval time is based on the refractory period after ejaculation of male rats. As a control group, female rats that were mated with vasectomized male rats to induce artificial pseudopregnancy were used.

[0078] (4) Fertilized egg transplantation As Example 1, 12 hours after artificially inducing pseudopregnancy with a pseudopregnancy inducer, fertilized eggs that had developed to the two-cell stage were transplanted into the oviducts of pseudopregnant female rats.

[0079] Also, as Example 2, 12 hours after artificially inducing pseudopregnancy with a pseudopregnancy inducer, cryopreserved fertilized eggs at the two-cell stage were transplanted into the oviducts of pseudopregnant female rats.

[0080] Furthermore, as Example 3, 24 hours after artificially inducing pseudopregnancy with a pseudopregnancy inducer, pronuclear-stage fertilized eggs were transplanted into the oviducts of pseudopregnant female rats.

[0081] Note that as Control Group 1, 12 hours after mating with a vasectomized male rat, pseudopregnancy was induced and fertilized eggs that had developed to the two-cell stage were transplanted into the oviducts of pseudopregnant female rats. Also, as Control Group 2, 12 hours after mating with a vasectomized male rat, cryopreserved fertilized eggs at the two-cell stage were transplanted into the oviducts of pseudopregnant female rats.

[0082] (5) Data analysis All data were subjected to a multiple comparison test using Ryan's method after a chi-square test.

[0083] For such pseudopregnant female rats in Examples 1 to 3 and Control Groups 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.

[0084]

Table 1

[0085] As shown in Table 1, in Example 1, among the transplanted fresh two-cell stage fertilized eggs, 63% implanted, and normal offspring were obtained from 54% of the fertilized eggs. In Example 2, among the transplanted frozen two-cell stage fertilized eggs, 46% implanted, and normal offspring were obtained from 24% of the fertilized eggs. In Example 3, among the transplanted fresh pronuclear stage fertilized eggs, 73% implanted, and normal offspring were obtained from 67% of the fertilized eggs.

[0086] In addition, in female rats induced to have artificial pseudopregnancy by mating with vasectomized male rats in Control Group 1, 72% of the transplanted fresh two-cell stage fertilized eggs implanted, and normal offspring were obtained from 63% of the fertilized eggs.

[0087] Also, in female rats induced to have artificial pseudopregnancy by mating with vasectomized 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. From the comparison with the results of these Control Groups 1 and 2, it was confirmed that in the female rats induced to have artificial pseudopregnancy in the examples, normal offspring could be obtained at a rate comparable to the conventional method.

[0088] <Example B: Comparison of Implantation Rate and Proportion of Normal Offspring by Stimulation Method> As Example 4, fresh two-cell stage fertilized eggs were transplanted into the oviducts of female rats induced to have pseudopregnancy in the same manner as in Example 1, except that the presentation pattern of vibration stimulation to the cervix was set to once for 120 seconds.

[0089] Also, as Example 5, fresh two-cell stage fertilized eggs were transplanted into the oviducts of female rats induced to have pseudopregnancy in the same manner as in Example 1, except that the presentation pattern of vibration stimulation to the cervix was set to twice for 30 seconds each, and an interval of 30 seconds was provided between the stimulations.

[0090] As Example 6, fresh pronuclear stage fertilized eggs were transplanted into the oviducts of female rats induced to have pseudopregnancy in the same manner as in Example 3, except that the presentation pattern of vibration stimulation to the cervix was set to twice for 30 seconds each, and an interval of 30 seconds was provided between the stimulations.

[0091] As Example 7, the presentation pattern of vibratory stimulation to the cervix was set to a total of 2 times for 30 seconds each, with an interval of 30 seconds between the stimulations. Ninety-six hours after artificially inducing pseudopregnancy with a pseudopregnancy inducer, fresh morula embryos were transplanted into the oviducts of pseudopregnant female rats in the same manner as in Example 1.

[0092] Regarding the pseudopregnant female rats of Examples 4 to 7 as described above, 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.

[0093]

Table 2

[0094] 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 morula embryos implanted, and normal offspring were obtained from 21% of the fertilized eggs.

[0095] <Example C: Comparison of Implantation Rate and Proportion of Normal Offspring by Stimulation Presentation to the Cervix on the Day of Fertilized Egg (Embryo) Transplantation> As Example 8, fresh fertilized eggs at the two-cell stage 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 before.

[0096] Also, as Example 9, fresh fertilized eggs at 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) transplantation, specifically 5 to 6 hours before.

[0097] For such pseudo-pregnant induced 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.

[0098]

Table 3

[0099] As shown in Table 3, in Example 8, among the fresh fertilized eggs at the two-cell stage that were transplanted, 75% implanted, and normal offspring were obtained from 68% of the fertilized eggs. Also, in Example 9, among the fresh fertilized eggs at the pronuclear stage that were transplanted, 72% implanted, and normal offspring were obtained from 67% of the fertilized eggs.

[0100] Therefore, it was confirmed that in the method for inducing artificial pseudo-pregnancy in mammals and the method for producing offspring from transplanted fertilized eggs in pseudo-pregnant animals of the present invention, normal offspring can be obtained with a success rate equal to or higher than that of conventional methods. In particular, in mammals to which vibration stimulation was applied to the uterine cervix on the day of fertilized egg (embryo) transplantation, it was confirmed that the implantation rate of the fertilized eggs exceeded 70%, and normal offspring were obtained from 65% or more of the fertilized eggs. Also, even when pronuclear stage fertilized eggs and morulae, which had a low success rate conventionally, were transplanted, it was confirmed that normal offspring were obtained at a rate almost comparable to that of the conventional method.

Explanation of Reference Signs

[0101] 1 Pseudo-pregnancy inducer 2 Vibration generator 3 Motor housing part 31 Spacer 32 Eccentric weight 4 Main body part 4a Rear surface 4b Front surface 41 Tip part 42 Outer case 43 Insertion member attachment shaft cylinder 43a Outer peripheral surface 44 Through hole 45 Switch member 46 Battery housing part 46a Anode terminal fitting 46b Cathode terminal spring 47 Barrel part 48 Substrate fixing part 49 Printed wiring board 5, 5’ Insertion member 5a Contact end part 51 Base end part 51a Lower end face 52 Fitting hole 52a Inner peripheral surface 53 Tip 54 Barrel part B Battery M Motor O o-ring S1, S2 Space SW Switch

Claims

1. A vibration generating device comprising a motor with an eccentric weight, a main body having a motor housing portion for housing the motor, a detachable insertion member at the tip of the main body and insertable into the vagina of a mammal, comprising: the insertion member is an elastic body, the tip of the elastic body is provided with a hemispherical metal contact end, the vibration generated by the motor is 18,000 to 25,000 times per minute, A false pregnancy inducer for producing offspring from transplanted fertilized eggs of a mammal.

2. The stimulation by the vibration emitted from the vibration generating device repeats the stimulation of 30 seconds once for 2 to 7 times, and a 30-second to 5-minute interval is interposed between the stimulations. The false pregnancy inducer for producing offspring from transplanted fertilized eggs of a mammal according to Claim 1.

3. the mammal is an anestrous cyclic animal, The false pregnancy inducer for producing offspring from transplanted fertilized eggs of a mammal according to Claim 1.

4. the anestrous cyclic animal is one selected from the group consisting of Rodentia, Lagomorpha, and Insectivora, The false pregnancy inducer for producing offspring from transplanted fertilized eggs of a mammal according to Claim 3.

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