CN111876374A - High-survival-rate sheep fertilized egg culture method for in-vitro culture of Hu sheep embryo - Google Patents
High-survival-rate sheep fertilized egg culture method for in-vitro culture of Hu sheep embryo Download PDFInfo
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Abstract
The invention discloses a method for culturing high-survival-rate sheep fertilized eggs for in-vitro culture of Hu sheep embryos, which comprises the following steps: (1) collecting oocytes: sucking egg cells from the ovary of the Hu sheep; (2) collecting sperms: extracting sperm from male Hu sheep; (3) fertilization: artificial fertilization is carried out on the ovum and the sperm; (4) embryo culture: putting the fertilized eggs into an incubator for culture; (5) and (4) observation: observing the development condition of the embryo; the incubator in the step (4) comprises a main body, a feeding device, a rotating frame, a culture device and a discharging device; the feeding device comprises a feeding channel, a conveying mechanism, a feeding hole, a rotating table and a transition mechanism; the method improves the survival rate of the embryo through artificial fertilization; the transition mechanism isolates the embryos from the outside air when the embryos are placed in the incubator, so that the development of the embryos is prevented from being influenced; the independent culture dish of getting is realized to discharging device, improves the embryonic survival rate.
Description
Technical Field
The invention belongs to the technical field of in vitro culture of sheep embryos, and particularly relates to a high-survival-rate sheep fertilized egg culture method for in vitro culture of Hu sheep embryos.
Background
With the development of modern reproduction biotechnology and the gradual improvement of embryo in vitro production technology, the sheep embryo in vitro production technology becomes an important way for accelerating variety breeding and excellent population propagation speed; although live offspring have been obtained by embryo in vitro production technology, the in vitro embryos obtained by in vitro fertilization generally have development blocking phenomenon in the culture process, so that the blastocyst development rate of the in vitro embryos is far lower than that of the in vivo embryos; the current nutrition environment for the Hu sheep embryo in vitro culture is one of the key reasons for the problems, for example, in the process of checking the fertilized ova of the sheep, the fertilized ova need to be taken out from the incubator, and the temperature, the humidity, the oxygen, the carbon dioxide and the like in the incubator are greatly influenced in the taking process.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the method for culturing the sheep fertilized eggs with high survival rate for the in-vitro culture of the Hu sheep embryos, wherein the taking operation does not easily influence the environment in the box.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for culturing high-survival-rate sheep fertilized eggs for the in-vitro culture of Hu sheep embryos comprises the following steps:
(1) collection of oocytes: directly sucking the oocytes from the ovaries of the Hu sheep by means of an ultrasonic detector, an endoscope or a laparoscope;
(2) collecting sperms: discharging sperm of the Hu sheep by an electrical stimulation method;
(3) fertilization: placing the egg cells and the sperms in a culture dish for artificial fertilization;
(4) embryo culture: putting the culture dish into an incubator for culture;
(5) and (4) observation: the culture solution is required to be replaced every 48-72 hours during the culture process of the embryo, and the development condition of the embryo is observed at the same time
The incubator in the step (4) comprises a main body, a feeding device arranged on the main body, a rotating frame arranged in the main body, a culture device arranged on the rotating frame, and a discharging device arranged on the rotating frame; the feeding device comprises a feeding channel arranged on the main body, a conveying mechanism arranged in the feeding channel, a feeding hole arranged at one end of the feeding channel, a rotating table arranged in the feeding channel and a transition mechanism arranged on the rotating table; firstly, placing a culture dish on a rotating table through a feeding hole, and then conveying the culture dish to a conveying mechanism through a transition mechanism; opening the conveying mechanism to convey the culture dish into the culture device on the rotating frame; when needing to observe or change the culture solution, rotate the swivel mount and make the culture dish enter into discharging device on, take out this culture dish alone and handle.
Through the mode of artificial fertilization, the screening of the Hu sheep egg cells and sperms is realized firstly; performing artificial fertilization on the activated egg cells and sperms; on one hand, the fertilization probability is improved; on the other hand, the fertilized embryo is healthier; the culture dish in the incubator can automatically rotate through the arrangement of the rotating frame, the manual position replacement is avoided, the influence of injured bacteria on embryos is prevented, and the survival rate is improved; the arrangement of the transition device realizes that the feed port is sealed when the culture dish is placed in the incubator, so that external air is prevented from entering the incubator during feeding, the environment in the incubator is prevented from being influenced by the external environment, the embryo development is influenced, and the survival rate of culture is improved; realize automatic entering into the incubator with the culture dish through transport mechanism's setting, the artificial contact of avoiding has reduced the risk of external bacterium to the embryo influence, has improved the survival rate.
In the step (3), the collected egg cells are selected and washed in a solid microscope, and then the egg cells are put into a culture solution with a ratio of 3-10 microliter for fertilization.
The method realizes the screening of the ovum and the sperm, and can remove some ovum and sperm with low activity; thereby improving the health of the fertilized embryo.
In the step (4), the culture environment in the incubator is air with culture conditions of 39 ℃, 100% humidity and 5% carbon dioxide.
The environment in the incubator is adapted to the growth of the Hu sheep embryo by the method, so that the embryo growth is improved; so that the embryo can develop smoothly.
The transition mechanism comprises two sealing plates arranged on the rotating platform, a rotating rod arranged on the rotating platform, a fixed clamp arranged on the rotating rod, a movable clamp arranged on one side of the fixed clamp, a first torsion spring arranged on the movable clamp, a positioning block arranged on the inner wall of the feeding channel, a first motor arranged on the rotating platform, a limiting block arranged on the rotating platform and a limiting spring arranged on the limiting block; the limiting block is moved to be abutted against the culture dish by the culture dish abutting against the limiting block; then the culture dish is fixed by the extrusion force of the fixed clamp and the movable clamp; starting a first motor and rotating the rotating platform; at the moment, the culture dish starts to rotate along with the culture dish, and then the feeding hole is sealed under the rotation of the sealing plate; the rotating table continues to rotate, the movable clamp is abutted against the positioning block, and the movable clamp is driven to rotate and deform; at this moment, under the action of the limiting spring, the limiting block is driven to move, so that the culture dish is driven to the conveying mechanism.
The sealing of the conveying mechanism and the feeding hole is realized through the arrangement of the sealing plate, and the outside air is prevented from entering the incubator when the rotating platform rotates; the environment in the incubator is not influenced by the outside, so that the embryo is not changed in the environment enough, and the survival rate is improved; the culture dish can automatically enter the conveying mechanism through the arrangement of the limiting block, so that the stability is improved; the embryo can not be affected when moving, and the injury is prevented; the culture dish is fixed through the arrangement of the fixed clamp and the movable clamp, and the culture dish is prevented from being separated from the rotating table when moving; the stability is improved, so that the embryo is not affected by injury; the positioning block is arranged to realize the propping with the movable clamp, so that the culture dish can be driven by the limiting block to enter the conveying mechanism; on one hand, the rotating platform is limited, so that the culture dish can accurately enter the conveying mechanism; on the other hand can be that the activity presss from both sides takes place deformation, prevents that the culture dish card from on transport mechanism.
The conveying mechanism comprises a conveying belt arranged in the feeding channel, a pushing plate arranged on the conveying belt, a second torsion spring arranged on the pushing plate and a spring piece arranged at one end of the conveying belt; when the culture dish moves to the conveyer belt, the culture dish firstly collides with the pushing plate to rotate anticlockwise, and other pushing plates which do not rotate collide with the culture dish; starting the conveyor belt, and then enabling the culture dish to abut against the spring piece under the driving of the conveyor belt; at the moment, the pushing plate pushes the culture dish to advance to drive the spring piece to deform; when the culture dish enters the culture device, the spring piece resets, and the culture dish is pushed to the culture device;
the automatic transmission of the culture dish is realized through the arrangement of the conveyor belt, so that the culture dish can be stably transmitted into the culture device; on one hand, automatic transmission is realized, the contact of bacteria on a culture dish is reduced, and the survival rate of embryos is improved; on the other hand, the transfer is stable, so that the shaking of the culture can be reduced, and the development of the embryo can be improved; the culture dish is pushed by the pushing plate, so that the culture dish is prevented from being clamped on the conveying belt under the influence of friction force, and the stability is improved; the movement of the culture dish is further pushed by the arrangement of the spring piece, so that the culture dish can automatically enter the culture device after the pushing of the conveying belt is lost; the stability is improved.
The culture device comprises a culture hole arranged on the rotating frame, a buffer ring arranged at the upper end of the culture hole, a bottom plate arranged in the culture hole, a separating plate arranged on the bottom plate, a first spring arranged on the bottom plate, an air inlet hole arranged at the bottom of the culture hole, a V-shaped rod arranged on the bottom plate, an elastic band arranged in the V-shaped rod and a pressing assembly arranged below the culture hole; starting the pressure applying assembly to drive the bottom plate to move upwards, wherein the culture dish enters the culture hole under the pressing action of the spring piece, and the culture dish is stably dropped on the separation plate under the action of the buffer ring; then the rotating frame is rotated to enable the culture hole to be far away from the pressure applying assembly; at the moment, under the action of the first spring, the bottom plate starts to move downwards; in the moving process, the V-shaped rod is driven to be folded, the bottom plate is slowly descended and reset by the tension of the elastic band in the folding process, and the culture dish is placed in the culture hole.
The culture dish can be stably fixed in the hole through the arrangement of the culture hole, so that the culture dish is kept relatively static in the main body; the influence on the development of the embryo during movement is prevented; the buffer ring is arranged to prevent the culture dish from colliding with the culture hole under the driving of the spring piece, so that the culture dish stably falls into the culture hole, and the stability is improved; the arrangement of the V-shaped rod realizes the combination with the lifting of the bottom plate, and the reset of the bottom plate is buffered; prevent the development of the embryo from being influenced by the violent movement of the bottom plate; the V-shaped rod is buffered by the elastic band, so that the stability is improved; the division with the culture dish has been realized through the setting of division board, prevents that the outside air from entering into in the incubator when taking out the culture dish.
The pressure applying assembly comprises a positioning groove arranged at the bottom of the culture hole, an inflator arranged at the bottom of the main body, a positioning ring arranged on the inflator and a second spring arranged on the positioning ring; when the rotating frame starts to rotate, the positioning ring is embedded with the positioning groove; at the moment, the inflator is butted with the air inlet; the aerator is activated to start pressing the culture well, and the bottom plate starts moving upwards.
The positioning groove and the positioning ring are arranged, so that the inflator can accurately abut against the culture hole after the rotating frame rotates; the separating plate is prevented from being unstable due to air leakage during pressure application, the stability is improved, and development damage caused by transitional bumping of embryos is prevented; the pressure on the culture holes is realized through the arrangement of the aerator, and the lifting stability of the bottom plate is improved after the culture holes are fully filled; on the other hand, the influence of the extra driving on the internal environment of the incubator is reduced.
The discharging device comprises a discharging barrel arranged on the rotating frame, a fixing frame arranged at the upper end of the discharging barrel, a fixed telescopic barrel arranged on the wall of the discharging barrel, a telescopic block arranged in the telescopic barrel, a rotating rod arranged on the telescopic block, a support arranged on the rotating rod, a fixing mechanism arranged on the support, a flip cover arranged on the discharging barrel and a driving block arranged on the flip cover; when the culture dish is required to be taken out, the rotating frame is rotated, and the culture dish is rotated to the discharging barrel; then the inflator is started again to drive the bottom plate to move upwards, at the moment, the separation plate moves upwards to abut against the bracket, and the bracket is driven to rotate; then under the action of the fixing mechanism, the bracket stably supports the culture dish; then the turnover cover is lifted, and at the moment, the driving block moves in the telescopic cylinder to drive the telescopic block to move upwards and drive the culture dish to move upwards; then the separation plate is propped against the fixed frame to realize the limit.
The discharging barrel is arranged to realize that the discharged material can be separated from the incubator, so that the external air is prevented from flowing into the incubator to influence the environment in the incubator during discharging; the telescopic cylinder is arranged, so that the influence on the inside of the incubator can be reduced when the telescopic block and the driving block move, and the stability of the environment in the incubator is improved; the support of the separation plate is realized through the arrangement of the bracket, so that the culture dish can be far away from the culture hole, and the independent discharge is realized; the separation plate is fixed through the fixing frame, so that on one hand, the discharge cylinder is sealed through the separation plate, and outside air is prevented from entering the incubator; on the other hand, the culture dish is made by the separating plate, so that the culture dish is prevented from being stressed unevenly and overturned when the culture dish is offset with the supporting block.
The fixing mechanism comprises a ratchet ring arranged on the telescopic block, a ratchet gear arranged on the rotary rod, a plum blossom groove arranged on the telescopic block, a counter rod arranged on the rotary block, a telescopic ball arranged on the counter rod and a telescopic spring arranged on the telescopic ball; when the culture dish moves upwards and abuts against the bracket, the rotating stick drives the ratchet gear to be matched with the ratchet ring; then the counter stick is driven to rotate, and the telescopic ball moves on the plum blossom groove; under the influence of the plum blossom groove, the rotating stick rotates, and the bracket in the rotating stick rotates to support the bottom of the culture dish.
The unidirectional rotation of the rotary rod is realized through the arrangement of the ratchet ring and the ratchet gear, so that the stability of the support frame is improved; the culture dish is prevented from falling; the rotary rod can automatically rotate after rotating for a certain angle through the arrangement of the mutual matching of the quincuncial grooves and the telescopic balls, so that the support frame can accurately abut against the bottom of the separation plate; the stability is improved, and the transition bump of the culture dish is prevented; thereby improving the safety of the embryo and further improving the survival rate.
In conclusion, the invention has the following advantages: according to the method, the ovum and the sperm of the Hu sheep are screened manually, and then artificial fertilization is performed, so that the vitality of the embryo is improved, and the survival rate of the embryo is improved; then, through the arrangement of the transition mechanism, when the culture dish enters the incubator, the outside air is reduced from entering the incubator; thereby make the environment in the incubator can not receive the influence, further improved embryo development's environment, improved the survival rate.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
Fig. 2 is a top view of the present invention.
Fig. 3 is a front view of the present invention.
Fig. 4 is a cross-sectional perspective view along a-a of fig. 2 of the present invention.
Fig. 5 is a cross-sectional view taken along B-B of fig. 3 of the present invention.
Fig. 6 is a cross-sectional view taken along C-C of fig. 2 of the present invention.
Fig. 7 is a partial view of the invention at F in fig. 5.
Fig. 8 is a partial view of the invention at E of fig. 5.
Fig. 9 is a partial view of the invention at B in fig. 4.
Fig. 10 is a partial cross-sectional view at D of fig. 5 of the present invention.
FIG. 11 is a partial view taken at A of FIG. 4 in accordance with the present invention.
Fig. 12 is a partial enlarged view of the invention at C in fig. 11.
Fig. 13 is a partial view of the invention at G in fig. 6.
FIG. 14 is a partial view taken at H of FIG. 5 in accordance with the present invention.
FIG. 15 is an enlarged view of a portion of FIG. 14 taken at J in accordance with the present invention.
Fig. 16 is an enlarged view of a portion of the invention at K in fig. 14.
Detailed Description
Example one
A method for culturing high-survival-rate sheep fertilized eggs for the in-vitro culture of Hu sheep embryos comprises the following steps:
(1) collection of oocytes: directly sucking the oocytes from the ovaries of the Hu sheep by means of an ultrasonic detector, an endoscope or a laparoscope;
(2) collecting sperms: discharging sperm of the Hu sheep by an electrical stimulation method;
(3) fertilization: placing the egg cells and the sperms in a culture dish for artificial fertilization;
(4) embryo culture: putting the culture dish into an incubator for culture;
(5) and (4) observation: replacing the culture solution every 48 hours during the culture process of the embryo, and observing the development condition of the embryo;
in the step (3), the collected egg cells are selected and washed in a solid microscope, and then the egg cells are put into a culture solution for fertilization, wherein the proportion of the culture solution is 3 microliters for one egg cell.
In the step (4), the culture environment in the incubator is air with culture conditions of 39 ℃, 100% humidity and 5% carbon dioxide.
As shown in fig. 1 to 16, the incubator in the step (4) comprises a main body 1, a feeding device 2, a rotating frame 3, a discharging device 4 and a culture device 5; the feeding device 2 comprises a feeding channel 21, a conveying mechanism 6, a feeding hole 22, a rotating table 23 and a transition mechanism 7; the feeding device 2 is arranged on the main body 1; the rotating frame 3 is rotatably arranged at the bottom of the main body 1; the discharging device 4 is arranged on the rotating frame 3; the culture device 5 is arranged on the rotating frame 3; the feeding channel 21 is arranged on the side wall of the main body 1; the conveying mechanism 6 is arranged in the feeding channel 21; the feed inlet 22 is arranged at one end of the feed channel 21; the rotating table 23 is rotatably embedded at the bottom of the feeding channel 21; the transition mechanism 7 is arranged on the rotating platform 23.
As shown in fig. 7-9, the transition mechanism 7 includes a sealing plate 71, a rotating rod 72, a fixing clip 73, a movable clip 74, a first torsion spring 75, a positioning block 76, a first motor 77, a limiting block 78, and a limiting spring 79; the sealing plates 71 are 2 and are arranged on the rotating platform 23; the rotating rod 72 is rotatably embedded on the feeding channel 21; the fixing clip 73 is arranged on the rotating stick 72; the movable clamp 74 is rotatably embedded on the rotary rod 72; the first torsion spring 75 is connected with the movable clamp 74 and the rotary rod 72; the positioning block 76 is arranged on the feeding channel 21 and can be abutted against the movable clamp 74; the first motor 77 is arranged on the rotating platform 23 and connected with the rotating rod 72; the first motor 77 is commercially available; the limiting block 78 is movably embedded on the rotating platform 23; the limit spring 79 connects the limit block 78 and the rotating table 23.
As shown in fig. 5 and 10, the conveying mechanism 6 comprises a conveying belt 61, a pushing plate 62, a second torsion spring 63 and a spring piece 64; the conveyor belt 61 is arranged in the feeding channel 21; the longitudinal section of the pushing plate 62 is 7-shaped and is rotatably embedded on the conveyor belt 61; the second torsion spring is connected with the pushing plate 62 and the conveyor belt 61; the spring plate 64 is provided at one end of the main body 1 at the front of the belt 61.
As shown in fig. 4, 11 and 12, the culture device 5 comprises a culture hole 51, a buffer ring 52, a bottom plate 53, a separation plate 54, a first spring 55, an air inlet hole 56, a V-shaped rod 57, an elastic band 58 and a pressing component 8; the culture holes 51 are arranged in the plurality of the culture holes and are uniformly distributed along the circumferential direction of the rotating frame 3; the buffer ring 52 is arranged at the port of the culture hole 51; the bottom plate 53 can be embedded in the culture hole 51 in an up-and-down moving way; the separating plate 54 is arranged on the bottom plate 53 and is connected in a clamping manner; the first spring 55 is connected with the bottom plate 53 and the culture hole 51; the air inlet 56 is arranged at the bottom of the culture hole 51; the V-shaped rod 57 is arranged at the bottom of the connecting bottom plate 53 and the culture hole 51; the elastic band 58 is embedded in the V-shaped rod 57; the pressurizing assembly 8 is disposed below the culture well 51.
As shown in fig. 4 and 11, the pressing assembly 8 includes a positioning groove 81, an inflator 82, a positioning ring 83, and a second spring 84; the positioning groove 81 is arranged at the bottom of the culture hole 51 and is annularly arranged at the bottom of the culture hole 51; the inflator 82 is arranged at the bottom of the main body 1, and the inflator 82 is directly purchased from the market; the positioning ring 83 is movably embedded on the inflator 82, and the positioning ring 83 can be abutted against the positioning ring 83; the second spring 84 connects the retaining ring 83 and the inflator 82.
As shown in fig. 13-14, the discharging device 4 includes a discharging barrel 41, a fixing frame 42, a telescopic barrel 43, a telescopic block 44, a rotating stick 45, a bracket 46, a fixing mechanism 9, a flip cover 47, and a driving block 48; the discharging barrel 41 is arranged on the rotating frame 3 and fixedly arranged at the upper end of the main body 1; the fixed frame 42 is arranged on the inner wall of the discharge cylinder 41; the telescopic cylinders 43 are 3 groups and are arranged on the outer wall of the discharge cylinder 41 along the circumferential direction of the discharge cylinder 41; the telescopic block 44 can be embedded in the telescopic cylinder 43 in an up-and-down moving way; the rotary rod 45 can be rotatably embedded on the telescopic block 44; the cross section of the bracket 46 is triangular and is arranged on the rotary rod 45; the fixing mechanism 9 is arranged on the bracket 46; the flip cover 47 is arranged on the discharging barrel 41; the driving block 48 is movably embedded in the telescopic cylinder 43 and is connected with the telescopic block 44.
As shown in fig. 14-16, the fixing mechanism 9 includes a ratchet ring 91, a ratchet gear 92, a plum blossom groove 93, an opposing stick 94, a telescopic ball 95, and a telescopic spring 96; the ratchet ring 91 is arranged on the telescopic block 44; the ratchet gear 92 is arranged on the rotary rod 45 and can be matched with the ratchet ring 91; the plum-blossom-shaped spline 93 is arranged in the telescopic block 44, and the cross section of the plum-blossom-shaped spline 93 is plum blossom-shaped; the counter roller 94 is arranged on the rotary roller 45; the telescopic ball 95 is telescopically embedded in the abutting stick 94 and can abut against the plum blossom key 93; the expansion spring 96 connects the counter stick 94 and the expansion ball 95.
The specific working process is as follows: the culture dish is conveyed to a conveyor belt 61 through a transition mechanism 7; then, the culture dish is conveyed into the culture hole 51 through the conveyor belt 61 for culture; when the culture dish needs to be taken out, the inflator is started to enable the separation plate frame to be arranged on the bracket; the flip cover is pulled, so that the culture dish moves upwards until the end part of the discharging barrel.
Example two
A method for culturing high-survival-rate sheep fertilized eggs for the in-vitro culture of Hu sheep embryos comprises the following steps:
(1) collection of oocytes: directly sucking the oocytes from the ovaries of the Hu sheep by means of an ultrasonic detector, an endoscope or a laparoscope;
(2) collecting sperms: discharging sperm of the Hu sheep by an electrical stimulation method;
(3) fertilization: placing the egg cells and the sperms in a culture dish for artificial fertilization;
(4) embryo culture: putting the culture dish into an incubator for culture;
(5) and (4) observation: replacing the culture solution every 72 hours in the culture process of the embryo, and observing the development condition of the embryo;
in the step (3), the collected egg cells are selected and washed in a solid microscope, and then the egg cells are put into a culture solution for fertilization, wherein the proportion of the culture solution is 3 microliters for one egg cell.
In the step (4), the culture environment in the incubator is air with culture conditions of 39 ℃, 100% humidity and 5% carbon dioxide.
EXAMPLE III
A method for culturing high-survival-rate sheep fertilized eggs for the in-vitro culture of Hu sheep embryos comprises the following steps:
(1) collection of oocytes: directly sucking the oocytes from the ovaries of the Hu sheep by means of an ultrasonic detector, an endoscope or a laparoscope;
(2) collecting sperms: discharging sperm of the Hu sheep by an electrical stimulation method;
(3) fertilization: placing the egg cells and the sperms in a culture dish for artificial fertilization;
(4) embryo culture: putting the culture dish into an incubator for culture;
(5) and (4) observation: replacing the culture solution every 60 hours in the culture process of the embryo, and observing the development condition of the embryo;
in the step (3), the collected egg cells are selected and washed in a solid microscope, and then the egg cells are put into a culture solution for fertilization, wherein the proportion of the culture solution is 3 microliters for one egg cell.
In the step (4), the culture environment in the incubator is air with culture conditions of 39 ℃, 100% humidity and 5% carbon dioxide.
Claims (9)
1. A method for culturing goat zygotes with high survival rate for the in vitro culture of Hu sheep embryos is characterized in that: the method comprises the following steps:
(1) collection of oocytes: directly sucking the oocytes from the ovaries of the Hu sheep by means of an ultrasonic detector, an endoscope or a laparoscope;
(2) collecting sperms: discharging sperm of the Hu sheep by an electrical stimulation method;
(3) fertilization: placing the egg cells and the sperms in a culture dish for artificial fertilization;
(4) embryo culture: putting the culture dish into an incubator for culture;
(5) and (4) observation: replacing the culture solution every 48-72 hours during the culture process of the embryo, and observing the development condition of the embryo;
the incubator in the step (4) comprises a main body (1), a feeding device (2) arranged on the main body (1), a rotating frame (3) arranged in the main body (1), a discharging device (4) arranged on the rotating frame (3) and a culture device (5) arranged on the rotating frame (3); the feeding device (2) comprises a feeding channel (21) arranged on the main body (1), a conveying mechanism (6) arranged in the feeding channel (21), a feeding hole (22) arranged at one end of the feeding channel (21), a rotating table (23) arranged in the feeding channel (21), and a transition mechanism (7) arranged on the rotating table (23); firstly, placing a culture dish on a rotating platform (23) through a feeding hole (22), and then conveying the culture dish to a conveying mechanism (6) through a transition mechanism (7); opening the conveying mechanism (6) to convey the culture dish into the culture device on the rotating frame (3); when the culture solution needs to be observed or replaced, the rotating frame (3) is rotated to enable the culture dish to enter the discharging device (4), and the culture dish is taken out independently for processing.
2. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 1, wherein: in the step (3), the collected egg cells are selected and washed in a solid microscope, and then the egg cells are put into a culture solution for fertilization, wherein the proportion of the culture solution is 3-10 microliter for one egg cell.
3. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 1, wherein: in the step (4), the culture environment in the incubator is air with culture conditions of 39 ℃, 100% humidity and 5% carbon dioxide.
4. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 1, wherein: the transition mechanism (7) comprises two sealing plates (71) arranged on the rotating platform (23), a rotating rod (72) arranged on the rotating platform (23), a fixing clamp (73) arranged on the rotating rod (72), a movable clamp (74) arranged on one side of the fixing clamp (73), a first torsion spring (75) arranged on the movable clamp (74), a positioning block (76) arranged on the inner wall of the feeding channel (21), a first motor (77) arranged on the rotating platform (23), a limiting block (78) arranged on the rotating platform (23), and a limiting spring (79) arranged on the limiting block (78); the limiting block (78) is pressed by the culture dish, so that the limiting block (78) moves to be abutted against the culture dish; then the culture dish is fixed by the extrusion force of the fixed clamp (73) and the movable clamp (74); starting a first motor (77) to rotate the rotating table (23); at the moment, the culture dish starts to rotate along with the culture dish, and then the feed port (22) is sealed under the rotation of the sealing plate (71); the rotating platform (23) continues to rotate, the movable clamp (74) is abutted against the positioning block (76), and the movable clamp is driven to rotate and deform; at the moment, under the action of a limiting spring (79), a driving limiting block (78) moves to drive the culture dish to the conveying mechanism (6).
5. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 1, wherein: the conveying mechanism (6) comprises a conveying belt (61) arranged in the feeding channel (21), a pushing plate (62) arranged on the conveying belt (61), a second torsion spring (63) arranged on the pushing plate (62), and a spring piece (64) arranged at one end of the conveying belt (61); when the culture dish moves to the conveyor belt (61), the culture dish firstly collides with the push plate (62) to rotate anticlockwise, and other non-rotating push plates (62) collide with the culture dish; starting the conveyor belt (61), and then enabling the culture dish to abut against the spring piece (64) under the driving of the conveyor belt (61); at the moment, the push plate (62) pushes the culture dish to advance to drive the spring piece (64) to deform; when the culture dish enters the culture device (5), the spring piece (64) resets to push the culture dish into the culture device (5).
6. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 1, wherein: the culture device (5) comprises a culture hole (51) arranged on the rotating frame (3), a buffer ring (52) arranged at the upper end of the culture hole (51), a bottom plate (53) arranged in the culture hole (51), a separation plate (54) arranged on the bottom plate (53), a first spring (55) arranged on the bottom plate (53), an air inlet hole (56) arranged at the bottom of the culture hole (51), a V-shaped rod (57) arranged on the bottom plate (53), an elastic band (58) arranged in the V-shaped rod (57), and a pressing assembly (8) arranged below the culture hole (51); starting the pressure applying assembly (8), driving the bottom plate (53) to move upwards, enabling the culture dish to enter the culture hole (51) under the pressing of the spring piece (64), and enabling the culture dish to stably fall on the separating plate (54) under the action of the buffer ring (52); then the rotating frame (3) is rotated to enable the culture hole to be far away from the pressure applying component (8); at the moment, under the action of the first spring (55), the bottom plate (53) starts to move downwards; during the movement, the V-shaped rod (57) is driven to be folded, and the tension of the elastic band (58) enables the bottom plate (53) to slowly descend and reset during the folding process, so that the culture dish is placed in the culture hole (51).
7. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 6, wherein: the pressing assembly (8) comprises a positioning groove (81) arranged at the bottom of the culture hole (51), an inflator (82) arranged at the bottom of the main body (1), a positioning ring (83) arranged on the inflator (82), and a second spring (84) arranged on the positioning ring (83); when the rotating frame (3) starts to rotate, the positioning ring (83) is embedded with the positioning groove (81); at the moment, the inflator (82) is butted with the air inlet hole (83); the inflator 82 is activated to start the pressure application to the inside of the culture well 51, and the bottom plate 53 starts to move upward.
8. The method for culturing the high-survival-rate sheep fertilized egg for the in-vitro culture of the Hu sheep embryo according to claim 1, wherein the discharging device (4) comprises a discharging barrel (41) arranged on the rotating frame (3), a fixing frame (42) arranged at the upper end of the discharging barrel (41), a fixed telescopic barrel (43) arranged on the wall of the discharging barrel (41), a telescopic block (44) arranged in the telescopic barrel (43), a rotating rod (45) arranged on the telescopic block (44), a bracket (46) arranged on the rotating rod (45), a fixing mechanism (9) arranged on the bracket (46), a turnover cover (47) arranged on the discharging barrel (41), and a driving block (48) arranged on the turnover cover (47); when the culture dish is required to be taken out, the rotating frame (3) is rotated, and the culture dish is rotated to the discharging barrel (41); then the inflator (82) is started again, the bottom plate (53) is driven to move upwards, the separating plate (54) moves upwards to abut against the bracket (46), and the bracket (46) is driven to rotate; then under the action of the fixing mechanism (9), the bracket (46) stably supports the culture dish; then the turnover cover (47) is lifted, at the moment, the driving block (48) moves in the telescopic cylinder (43), the telescopic block (44) is driven to move upwards, and the culture dish is driven to move upwards; then the separating plate (54) is abutted by the fixing frame (42) to realize the limit.
9. The method for culturing high-survival-rate sheep zygotes for in vitro culture of Hu sheep embryos according to claim 8, wherein: the fixing mechanism (9) comprises a ratchet ring (91) arranged on the telescopic block (44), a ratchet gear (92) arranged on the rotary rod (45), a quincunx groove (93) arranged on the telescopic block (44), a counter rod (94) arranged on the rotary rod (45), a telescopic ball (95) arranged on the counter rod (94), and a telescopic spring (96) arranged on the telescopic ball (95); when the culture dish moves upwards to abut against the bracket (46), the rotating stick (45) drives the ratchet gear (92) to be matched with the ratchet ring (91); then the counter rod (94) is driven to rotate, and at the moment, the telescopic ball (95) moves on the plum blossom groove (93); under the influence of the plum blossom groove (93), the rotating rod (45) rotates, and at the moment, the bracket (46) in the rotating rod (45) rotates to support the bottom of the separating plate (54).
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