CN211767665U - Insulation can that ox living body ovum collection was used - Google Patents
Insulation can that ox living body ovum collection was used Download PDFInfo
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- CN211767665U CN211767665U CN202020194797.4U CN202020194797U CN211767665U CN 211767665 U CN211767665 U CN 211767665U CN 202020194797 U CN202020194797 U CN 202020194797U CN 211767665 U CN211767665 U CN 211767665U
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Abstract
The application relates to an incubator for egg taking of a live cow. The insulation can that the living ox body ovum collection was used includes: the heat preservation core is a square calcium silicate sintered body, a plurality of test tube holes for accommodating egg taking test tubes are arranged on the top surface of the heat preservation core in a sunken mode, the interior of the calcium silicate sintered body is of a porous structure, the surface of the calcium silicate sintered body is glazed to seal the whole interior of the heat preservation core, and liquid energy storage agents are filled in pores in the interior of the calcium silicate sintered body; a case for accommodating a main body portion of the insulation core; and the box cover is used for covering the top of the heat preservation core and is integrated with the box body. The application of the incubator can realize that the temperature of liquid in the test tube is kept when the live cow egg is collected, and the purpose of maintaining the temperature in the process of transporting to a laboratory is realized, and the incubator is particularly suitable for the occasion of collecting the live cow egg with lower egg collecting environmental temperature.
Description
Technical Field
The application belongs to the technical field of biology, relates to a method for live ovum collection, and particularly relates to an incubator used in a live cow ovum collection process. The method and the used heat preservation box have the characteristics of good heat preservation effect and convenience in use, and the heat preservation box and the method provided by the application have important significance for improving the working efficiency of live cow egg collection.
Background
The embryo culture technology has important values for the breeding potential excavation, the rapid propagation and the germplasm resource protection of livestock. There are many factors that affect embryo in vitro culture and embryo in vitro preservation, among which oocyte in vitro maturation, in vitro fertilization, early embryo in vitro culture and embryo freezing are four major links in the in vitro embryo production process.
Bovine live body egg extraction (OPU) is a very practical embryo engineering technique for bovine embryo experimental manipulation and production and reproduction following In Vitro Fertilization (IVF) and Embryo Transfer (ET). This technique has been widely used for in vitro production of bovine embryos since the 20 th century, 80 s, reported the performance of bovine live body egg-taking operations under B-ultrasonic surveillance. The technology of taking the eggs from the live bodies of the cattle has the advantages of wide application, convenient operation, high efficiency, repeatability, small damage and the like. By combining the two technologies of live cow ovum taking and in vitro fertilization, the breeding potential of the improved cow can be fully improved, and high-quality embryos can be produced in large quantities.
The method for taking the bovine live body eggs provides a valuable method for producing high-quality bovine embryos in vitro. With the completion of the bovine genome sequencing and the drawing of new quantitative character sites, oocytes with clear genetic background are recovered by taking eggs from bovine living bodies, thereby providing valuable experimental materials for researching the bovine embryo development on the molecular level and finally generating remarkable economic and practical values for producing high-quality bovine embryos.
In Vitro Fertilization (In Vitro Fertilization) or (external Fertilization) refers to a technique In which sperm and eggs of a mammal complete a Fertilization process In an environment artificially controlled In Vitro, abbreviated In the english to IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother is called a test-tube animal. The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal breeding biotechnology.
The in vitro fertilization technology has important significance for animal reproductive mechanism research, livestock production, medicine, endangered animal protection and the like. For example, using mouse, rat or rabbit as experimental material, the in vitro fertilization technique can be used to study the gametogenesis, fertilization and early embryonic development mechanism of mammals. In the livestock breed improvement, the in vitro fertilization technology provides a cheap and efficient means for embryo production, and has important values for fully utilizing excellent breed resources, shortening the breeding cycle of livestock, accelerating the breed improvement speed and the like. In humans, IVF-ET technology is one of the important measures to treat certain infertility and to overcome sexual linked diseases. In vitro fertilization is also an indispensable component of modern biotechnology, such as mammalian embryo transplantation, cloning, transgenosis, sex control and the like.
With the development of modern agricultural science and technology, in order to make full use of the breeding potential of elite cows and accelerate the genetic breeding process, it becomes necessary to apply a new efficient breeding technology in production practice. In-vivo egg taking (OPU) and In Vitro Fertilization (IVF) are new embryo engineering technologies that have been developed rapidly In the eighties of the twentieth century, and a large number of embryos with definite genetic pedigrees can be obtained by combining the OPU and the IVF, so that the generation interval is shortened. At present, the two technologies become important breeding technologies adopted by farmers in animal husbandry developed countries such as europe, the united states and oceania for expanding stock cow groups. However, with the conventional bovine embryo culture system (CR1aa and SOF liquid), the blastocyst development rate of bovine in vitro fertilization is low, and the embryo quality is far inferior to that of in vivo embryos, so that the pregnancy rate after embryo transfer recipient is low, and therefore how to improve the blastocyst development rate and the embryo quality becomes the focus of in vitro fertilization embryo production and research.
As early as 1878, German Scnenk began to explore the in vitro fertilization technique of mammals using rabbits and guinea pigs as materials. However, in 1951, the in vitro fertilization technology has not been developed in a breakthrough after sperm capacitation was discovered by Zhang Xuanling and Austin, respectively. The cattle in vitro fertilization technology is influenced by the aspects of oocyte in vitro maturation, sperm in vitro capacitation, fertilized egg in vitro culture environment and the like.
An important tool for taking the eggs of the cattle living bodies is a B-ultrasonic diagnostic instrument for animals, which is designed according to the B-ultrasonic principle and is commonly used in human clinic. The B-ultrasonic diagnosis has important significance for improving the management level of the animal husbandry and promoting the economic development of the animal husbandry. The method for evaluating the reproductive capacity of the buffalo by using the B ultrasonic diagnostic technology for animals can enable people to know and know the states of the ovary and the uterus of the buffalo in the oestrus cycle and pregnancy, better improve the reproductive performance and promote the genetic process of animals.
In the case of a B-mode diagnostic apparatus, the collected follicles are kept at a predetermined temperature for a period of time, for example, at about 37 ℃ for several hours, before subsequent operations are performed, but under the usual conditions of live bovine ova aspiration, it is often difficult to achieve this temperature in the room, and even lower ambient temperatures are often encountered.
Because each time an ovum is taken to take an ovum next time, an ovum is taken from one cow to the next year, and a longer time is usually needed until a completely larger number of ova are taken, the ovum taken first and the ovum taken later have different placing time before the operations such as ova detection and the like are carried out in succession and uniformly, and the effect and the efficiency of taking the ova by living bodies are generally influenced by different waiting time lengths of the follicle in the ova-extracting liquid.
In particular, when the temperature of the follicular fluid is low at the outdoor temperature during the egg collection of the bovine OPU, the quality of the oocyte is affected by the temperature reduction of the follicular fluid.
Accordingly, it would be desirable to provide a holding device that maintains the temperature of the fluid in the tube while the OPU is being performed, and also maintains the temperature during transport to the laboratory.
SUMMERY OF THE UTILITY MODEL
The present application aims to provide a heat-insulating device which maintains the temperature of a liquid in a test tube while performing OPU, and maintains the temperature during transportation to a laboratory, and further expects that follicles collected from bovine living bodies will remain in a biological state unchanged after a sufficiently long period of time in an egg-extracting solution. The inventors have surprisingly found that one or more of the above objects can be completely achieved with the heat retention device of the present application. The present application has been completed based on this finding.
On the one hand, the application relates to an insulation can that ox living body ovum collection used, its characterized in that, it includes:
the heat preservation core is a square calcium silicate sintered body, a plurality of test tube holes for accommodating egg taking test tubes are arranged on the top surface of the heat preservation core in a sunken mode, the interior of the calcium silicate sintered body is of a porous structure, the surface of the calcium silicate sintered body is glazed to seal the whole interior of the heat preservation core, and liquid energy storage agents are filled in pores in the interior of the calcium silicate sintered body;
a case for accommodating a main body portion of the insulation core;
and the box cover is used for covering the top of the heat preservation core and is integrated with the box body.
The heat preservation box for the egg collection of the living cattle is characterized in that the calcium silicate sintered body is a cuboid.
The incubator for egg collection of living cattle according to the present application is characterized in that the calcium silicate sintered body is a cube.
The heat preservation box for egg collection of the living cattle is characterized in that the porosity inside the calcium silicate sintered body is 10-35%. In the context of the present application, the term "porosity" refers to the percentage of internal pore space of the calcium silicate in the volume of the entire sintered body after the sintered body is formed by the sintering process, as is well known in the art. In addition, calcium silicate is formed into a sintered body by a sintering process, which is also well known to those skilled in the art.
The heat preservation box for egg collection of the living cattle is characterized in that the porosity inside the calcium silicate sintered body is 15-30%.
The heat preservation box for egg collection of the living cattle is characterized in that the porosity inside the calcium silicate sintered body is 20-25%.
The heat preservation box for the ovum collection of the living cattle is characterized in that the test tube hole is used for accommodating an ovum collection test tube with the capacity of 10-100 ml.
The heat preservation box for the ovum collection of the living cattle is characterized in that the test tube hole is used for accommodating an ovum collection test tube with the capacity of 20-60 ml.
The heat preservation box for the live cow egg taking is characterized in that the depth of the test tube hole accounts for 60-90% of the height of the heat preservation core. The proportion of the depth of the test tube hole to the height of the heat preservation core refers to the proportion of the depth of the test tube hole penetrating into the heat preservation core, the proportion cannot be too small, the utilization rate of the heat preservation core is not high, the proportion cannot be too large, and the physical strength of the heat preservation core is affected.
The heat preservation box for the live cow egg taking is characterized in that the depth of the test tube hole accounts for 65-85% of the height of the heat preservation core.
The incubator for egg collection of the living cattle body is characterized in that the liquid energy storage agent is glycol.
According to the application, the incubator for the egg taking of the live cattle can provide required preservation temperature for the egg taking test tube within required time. For example, it can maintain the egg collecting tube temperature in a desired temperature range of 10 to 40 ℃ for a period of not less than 3 hours or not less than 5 hours.
The application second aspect provides a method of ox living body ovum pick-up, and it includes places the follicular fluid of extraction in the ovum pick-up test tube, the ovum pick-up test tube stay in this application first aspect the test tube hole of insulation can is downthehole, this insulation can be in the time of needs for the ovum pick-up test tube provides required save temperature.
Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.
All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.
In the present application, directional terms may be understood in conjunction with the description and drawings, for example, in describing an insulating core, the tube bore is oriented upward.
Drawings
FIG. 1 is a schematic view showing the overall structure of an incubator for collecting bovine eggs from a living body according to the present invention.
FIG. 2 is a schematic view of the heat-insulating core of the incubator for collecting bovine eggs from a living body according to the present invention.
The application relates to a heat preservation box for live cow egg taking, which is marked and gathered by main parts as follows: the test tube comprises a heat preservation core 1, a calcium silicate sintered body 2, a test tube hole 3, a liquid energy storage agent 4, a box body 5 and a box cover 6.
Detailed Description
The present application can be further described by the following examples, however, the scope of the present application is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the present application without departing from the spirit and scope of the application. The materials used in the tests and the test methods are generally and/or specifically described herein. Although many materials and methods of operation are known in the art for the purposes of this application, this application is nevertheless described herein in detail as far as possible. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The present application is further described with reference to the accompanying drawings. Fig. 1 is a schematic view of the overall structure of the incubator for living bovine ovum collection of the present application, and fig. 2 is a schematic view of the heat insulating core of the incubator for living bovine ovum collection of the present application.
As shown in fig. 1 and 2, the present application relates to an incubator for egg collection of a living cow, comprising:
the heat preservation core 1 is a square calcium silicate sintered body 2, a plurality of test tube holes 3 for accommodating egg taking test tubes are arranged on the top surface in a concave mode, the interior of the calcium silicate sintered body 2 is of a porous structure, the surface of the calcium silicate sintered body 2 is glazed to seal the whole interior of the heat preservation core 1, and liquid energy storage agents 4 are filled in pores in the interior of the calcium silicate sintered body 2;
a box body 5 for accommodating the main body part of the heat preservation core 1;
and a cover 6 for covering the top of the thermal insulation core 1 and integrally formed with the case 5.
As is well known, a sintered body formed by a sintering process of calcium silicate is a porous structure, and after the surface (including the surface of a pit of a test tube hole 3) of the sintered body is subjected to glazing, a sealing layer can be formed on the surface of the sintered body, so that the pore spaces in the whole sintered body are sealed; of course, in order to facilitate the filling liquid full-energy storage agent 4, a filling hole can be reserved before the surface of the whole body is glazed, and the filling hole is finally sealed, which is very easy to realize in industrial production.
According to a specific example of the incubator for egg collection from a living bovine body, the calcium silicate sintered body 2 is a rectangular parallelepiped.
According to a specific example of the incubator for egg collection from a living bovine body, the calcium silicate sintered body 2 is a cube.
According to a specific example of the incubator for egg collection of living bovine bodies, the porosity of the interior of the calcium silicate sintered body 2 is 10 to 35%. In the context of the present application, the term "porosity" refers to the percentage of internal pore space of the calcium silicate in the volume of the entire sintered body after the sintered body is formed by the sintering process, as is well known in the art. In addition, calcium silicate is formed into a sintered body by a sintering process, which is also well known to those skilled in the art.
According to a specific example of the incubator for egg collection of living bovine bodies, the porosity of the interior of the calcium silicate sintered body 2 is 15 to 30%.
According to a specific example of the incubator for egg collection of living bovine bodies, the porosity of the interior of the calcium silicate sintered body 2 is 20 to 25%.
According to a specific example of the incubator for ovum collection of the living bovine bodies, the test tube hole 3 is used for accommodating an ovum collection test tube with the capacity of 10-100 ml.
According to a specific example of the incubator for ovum collection of the living bovine bodies, the test tube hole 3 is used for accommodating an ovum collection test tube with the capacity of 20-60 ml.
According to a specific example of the heat preservation box for the live cow ovum collection, the depth of the test tube hole 3 accounts for 60-90% of the height of the heat preservation core 1. The proportion of the depth of the test tube hole 3 to the height of the heat preservation core 1 is the proportion of the depth of the test tube hole 3 penetrating into the heat preservation core 1, the proportion cannot be too small, the utilization rate of the heat preservation core 1 is not high, the proportion cannot be too large, and the physical strength of the heat preservation core 1 is affected.
According to a specific example of the heat preservation box for the live cow egg taking, the depth of the test tube hole 3 accounts for 65-85% of the height of the heat preservation core 1.
According to a specific example of the incubator for egg collection of a living bovine body, the liquid energy storage agent 4 is ethylene glycol.
According to a specific example of the incubator for egg taking of the living cattle, the incubator can provide a required preservation temperature for the egg taking test tube within a required time. For example, it can maintain the egg collecting tube temperature in a desired temperature range of 10 to 40 ℃ for a period of not less than 3 hours or not less than 5 hours.
In the present application, the box body 5 and the box cover 6 may be made of any suitable material, for example, paper, plastic, metal, glass, and combinations thereof, and especially, the material of plastic, metal, and combinations thereof is preferably used, and the material of the box body 5 and the box cover 6 is widely selected and does not affect the implementation of the present application.
The incubator of the present application can be used as follows: heating (for example, electrically heating, for example, by using an electric heater or an electric blanket) or cooling (for example, ice bath) the heat preservation core 1 (in which the ovum collection tube is not placed in advance) to a desired temperature by a conventional method (for example, heating or cooling the heat preservation core 1 to a desired temperature (for example, 38 ℃) within a range of 10-40 ℃), placing the heat preservation core 1 in the box body 5 and the box cover 6, opening the box cover 6 when in use, adding the collected follicles to the ovum collection tube which is placed in the tube hole 3 in advance and preheated/or pre-cooled to the desired temperature, and performing subsequent follicle processing operation after the collected follicles are left in the ovum collection tube for a certain time.
For example, with the heat preservation box of the present application, 35 test tube holes 3 with the length, the width and the height of 40cm 30cm of the heat preservation core 1 and the diameter of 2cm are uniformly arranged, the porosity of the interior of the calcium silicate sintered body 2 is 25%, the test tube capacity is 55ml, the egg collection test tube is filled with warm water with the temperature of 38 ℃, then the heat preservation core 1 is heated to the temperature of 38 ℃ in whole (the temperature is reached by the measurement in the test tube holes of each part of the heat preservation core 1), the heat preservation core is placed in a box body 5 and a box cover 6, the heat preservation time when the temperature of the water in the egg collection test tube is reduced to 35 ℃ is measured by standing in the environment with the temperature of 12 ℃; as a result, the holding time under the above-mentioned design conditions reached 5.3 hours. Therefore, the heat preservation box is particularly suitable for occasions with low egg picking environment temperature.
A typical method for collecting bovine oocytes from a living body by temporarily storing collected follicular fluid using the incubator described above is as follows: 4-8 year old cows with normal ovarian function through rectal examination are used as test cows for live egg collection. The egg taking device comprises: HS2000B ultrasonic imaging instrument manufactured by Fushiping corporation of Japan, Allocara vagina sector scanning probe, 60cm long ovum collecting needle, suction negative pressure device, constant temperature device of follicular fluid collecting tube, embryo filtering cup, etc. The formulation of the egg extract is set forth in the specific examples, which are sterilized by filtration. Collection of oocytes: fixing the tested cattle according to the embryo transplantation operating rules, removing the excrement of the rectum, and cleaning and disinfecting the trunk and the pudendum. Meanwhile, the B-mode ultrasonic instrument is unfolded and placed on an operation site. Putting the pre-packaged egg-extracting liquid (40 ml per cow) and 1 follicle-fluid collecting tube of 50ml into a thermostatic device at 38 ℃, taking out an egg-collecting needle and a connecting rubber tube from a disinfection bag, placing the egg-collecting needle in a clamping groove on a probe, connecting the other end of the rubber tube with the collecting tube, and wiping the collecting tube with 75% alcohol for standby. An operator stretches into the rectum with the left hand to fix the right ovary, slowly inserts the probe provided with the ovum pick-up needle into the vagina to be against the fornix of the vagina, places the probe on the right side of the fornix, draws the ovary to be tightly attached to the front end of the probe with the left hand, displays an ovary image on a monitor of a B-type ultrasonic instrument at the moment, turns the ovary back and forth and left and right, determines the number of follicles, adjusts the positions of the ovary and the probe to enable the follicles to be positioned on a puncture guide line, the operator pushes the ovum pick-up needle on the probe forward to puncture the follicles, simultaneously, a foot switch is used to start a vacuum pump, the pressure of 40-50 mm mercury column is generally adopted, the image of the sucked follicles can be reduced on the monitor, the shape becomes irregular until the follicular fluid is completely extracted, the follicle image disappears, the ovum pick-up needle exits, then punctures the 2 nd follicle, and the process is repeated, and then the ovary on the other side is changed to perform follicle puncture to aspirate follicular fluid by the same method. In the process of follicle suction, 1-2 follicles are sucked every time, the ovum suction liquid is used for flushing the ovum collection needle and the connecting rubber tube for 1 time, so that blood is prevented from being coagulated in the ovum suction needle, and the continuous puncture and recovery of oocytes are prevented from being influenced. Finally, a follicle fluid collecting tube (follicle is suspended in a mixed solution of follicle fluid and ova-extracting fluid, the volume ratio of the follicle fluid to the ova-extracting fluid is about 1: 8.5) is sent to a laboratory for ovum detection and classification within a specified time. Examination of oocytes: in a laboratory, the liquid in the follicular fluid collecting tube is poured into a filter cup, and the collecting tube is washed 2 times by using an egg washing liquid (the formula is shown in the operation examples cited later) at the temperature of 30-35 ℃, and is also poured into the filter cup. When the follicular fluid in the collecting tube is mixed with blood, the collecting tube is diluted by the ovum washing fluid until the follicular fluid is clear, then the fluid flows out of a filter screen on the wall of the cup, 10-15 ml of the fluid is left, the fluid is washed for 2 times by a HEPES maturation culture solution (the formula is shown in the operation examples cited later), and then the oocyte is checked under a stereoscope for classification. Grading the oocyte: the number of cumulus cell layers of Cumulus Oocyte Complexes (COCs) to be examined is classified into A, B two stages, in which the number of cumulus cell layers of the A stage is 3 and more than 3 (they are generally uniform in cytoplasm); b stage: cumulus cells have 2 or less than 2 layers (they are generally poorly cytoplasmic in homogeneity); grade a is available oocytes for culture. For the sample in one follicular fluid collection tube, the number of cumulus cell summation layers of 30 oocytes was calculated by the above-described examination method, and the number of cumulus cell summation layers was used as a typical state of the oocytes in the sample in the follicular fluid collection tube.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The utility model provides an insulation can that ox living body ovum collection used which characterized in that, it includes:
the heat preservation core is a square calcium silicate sintered body, a plurality of test tube holes for accommodating egg taking test tubes are arranged on the top surface of the heat preservation core in a sunken mode, the interior of the calcium silicate sintered body is of a porous structure, the surface of the calcium silicate sintered body is glazed to seal the whole interior of the heat preservation core, and liquid energy storage agents are filled in pores in the interior of the calcium silicate sintered body;
a case for accommodating a main body portion of the insulation core;
and the box cover is used for covering the top of the heat preservation core and is integrated with the box body.
2. The incubator according to claim 1, wherein the sintered calcium silicate is a rectangular parallelepiped or a cube.
3. The incubator according to claim 1, wherein the porosity of the inside of the sintered calcium silicate body is 10 to 35%.
4. The incubator according to claim 1, wherein the porosity of the inside of the sintered calcium silicate body is 15 to 30%.
5. The incubator according to claim 1, wherein the porosity of the inside of the sintered calcium silicate body is 20 to 25%.
6. The incubator according to claim 1, wherein the test tube hole is adapted to accommodate a test tube for collecting eggs having a volume of 10-100 ml.
7. The incubator according to claim 1, wherein the test tube hole is adapted to accommodate a 20-60 ml egg collection test tube.
8. The incubator according to claim 1, wherein the depth of the test tube hole is 60-90% of the height of the incubation core.
9. The incubator according to claim 1, wherein the depth of the test tube hole is 65-85% of the height of the incubation core.
10. The incubator of claim 1, wherein the liquid energy storage agent is ethylene glycol.
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