Apis cerana drone sperm extraction device and efficient extraction method

Abstract

The invention discloses a Chinese bee drone semen extraction device and an efficient extraction method, the semen extraction device comprises an extraction type piston, a drone imbedding device, a semen collection channel and a movable semen collection table, and the extraction type piston is arranged at an upper port of the drone imbedding device; a seminal fluid collecting channel is arranged below the drone imbedding device, and an EP pipe is arranged on a movable seminal fluid collecting table below the seminal fluid collecting channel and used for receiving extracted seminal fluid. The semen extraction and enrichment method comprises the following steps: placing a drone into the drone fixator, keeping the abdomen upward and enabling 2-3 back plates at the tail part of the drone to extend out of the fixing device, enabling the chest and the abdomen of the drone to directly face an electric shock opening, and then installing an extraction type piston to stabilize the position of the drone; and the chest and the abdomen of the drone are electrically shocked simultaneously by a 220V voltage contact, so that the cutin, the mucilage gland and the positive stem bulb of the drone are completely turned outwards, and then the sperm extraction by electric shock is completed. After drone electric shock operation is completed, a basic auxiliary buffer solution is used for flushing seminal fluid on the outer side of a positive stem ball subjected to electric shock extroversion, the seminal fluid flows into an EP tube, the seminal fluid collecting method guarantees the activity and the number of sperms, and impurities in the extracted seminal fluid are remarkably reduced; and then freezing and centrifuging to obtain the high-enrichment and high-activity seminal fluid.

Description

Technical Field

[0001] The present invention relates to an Apis cerana drone sperm extraction device and an efficient extraction method, belonging to the technical field of bee breeding. Background Technique

[0002] A bee colony consists of a queen bee, worker bees and drone bees. The drone bees are specialized in mating with virgin queen bees to provide paternal genetic information. The quality of the queen bee's mating affects the stability of the entire bee colony, which is closely related to the quantity and quality of sexually mature drone bees. However, there are often many uncontrollable factors in the natural mating of queen bees. For example, extreme weather, predation by natural enemies, interference between different bee species, etc. are likely to cause the failure of the queen bee's mating, resulting in serious consequences such as the collapse of the bee colony. Modern methods usually adopt the artificial insemination technology of queen bees to make up for this defect. To successfully implement this technology, in addition to cultivating well-developed virgin queen bees in advance, how to efficiently and cleanly extract the semen of sexually mature high-quality drone bees is the key factor determining the success or failure of insemination. The traditional method for extracting sperm from drone bees is to grab the drone bees that have reached the sexually mature age and press them to extract sperm. First, press their chests and abdomens. After the cuticle and the phallus bulb turn outwards, then use a queen bee insemination needle to suck the semen outside the phallus bulb and directly inject it (artificial insemination); however, since the strength of finger pressing is not easy to control, it may cause the semen to splash or the phallus bulb to rupture. Moreover, during the sperm extraction process, the injection needle may suck impurities such as mucus glands into the needle, contaminating the semen with body fluids; at the same time, the everted semen may also contaminate the operator's fingers or the body surface of the drone bees, which is extremely easy to waste and contaminate the semen, increasing the risk of the queen bee dying due to bacterial infection or blocked fallopian tubes.

[0003] Apis cerana (referred to as Chinese bee) is the general name of Apis cerana in China. The drone bees of Apis cerana are drone bees of Chinese bees. Compared with the drone bees of another reared bee species - Western honey bees, they are characterized by small size, less semen volume and high viscosity. For the improvement of bee germplasm, the artificial insemination technology is adopted, that is, collecting the semen of high-quality drone bees and injecting it into virgin queen bees. For the Western honey bee drone bees with large size and large semen volume, the pressing method can be used for artificial sperm extraction. However, when using the pressing method to extract sperm from Apis cerana drone bees and collecting semen with a traditional capillary tube, due to the pressing force problem, the semen often adheres to the body surface of the drone bees or the operator's fingers, resulting in contamination, or the semen is mixed with impurities such as mucus glands, and it is also easy to block the capillary tube when collecting semen, extremely easily causing problems of semen waste and semen contamination.

[0004] In the aspect of semen extraction from drones of Apis cerana cerana, since the drones of Apis cerana cerana are smaller in size compared to those of Western honey bees, the semen volume of a single drone is less and viscous, and the extracted semen usually contains substances such as mucus glands, resulting in semen contamination. At the same time, the individual size of the drones of Apis cerana cerana is too small to extract enough semen to fertilize a single queen bee at one time. A large number of drones need to be captured at one time to extract semen. In this process, how to quickly achieve batch collection of drone semen and avoid semen deterioration is also an urgent problem to be solved. Summary of the Invention

[0005] Aiming at the above deficiencies existing in the prior art, the purpose of the present invention is to provide a drone semen extraction device to solve the problems that the current technology for extracting and enriching semen from drones of Apis cerana cerana is not yet mature, there is no special extraction equipment for the semen of drones of Apis cerana cerana, the traditional method of finger pressing for semen extraction cannot achieve pollution-free and rapid semen extraction, and there are also great deficiencies in the utilization and application of high-quality semen for artificial insemination of queen bees.

[0006] Furthermore, the present invention also provides a method for efficiently extracting and enriching semen from drones of Apis cerana cerana using the above device, to solve the problems of low semen extraction efficiency, unstable quality, and semen contamination.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: The present invention provides a semen extraction device for drones of Apis cerana cerana, which includes a retractable piston, a drone inserter, and a semen collection channel, and also includes a sampling table, a movable semen collection table, and an EP tube; the retractable piston is arranged at the upper port of the drone inserter; the drone inserter is cylindrical and has a frustum shape. There are two sector-shaped hollow openings near the lower opening. The drone inserter is used to fix mature drones and cooperate with the retractable piston to extract semen from the drones; a semen collection channel is arranged below the drone inserter, and an EP tube is arranged below the semen collection channel to collect the extracted semen.

[0008] Furthermore, the drone inserter is movably arranged on the semen collection table and is set at an angle of 45°, which is convenient for electroejaculation operation.

[0009] Furthermore, the semen collection channel is a pipe groove structure with a wider upper part and a narrower lower part (the pipe groove is about 10 mm long and about 2 mm deep), which can prevent semen overflow during collection.

[0010] Furthermore, the retractable piston, the drone inserter, the semen collection channel, and the corresponding 10 groups of EP tubes are arranged on the semen collection table of the sampling table, and are designed in one row or multiple rows. Preferably, 5 - 20 groups are used to achieve efficient semen extraction.

[0011] The present invention also provides a method for efficiently extracting and enriching semen from drones of Apis cerana cerana, which includes steps such as drone cultivation, drone fixation and electroejaculation, drone semen collection, and drone semen enrichment; among them, Drone fixation and electroejaculation: Select drones at the mature age (16 - 22 days), place them in a drone fixator with their abdomens facing upward and the 2 - 3 dorsal plates of the tails extending out of the fixing device, so that the chest and abdomen of the drone face the electroshock port, and then install a retractable piston to stabilize the position of the drone; for sperm extraction, use a storage type AC variable frequency device, and simultaneously electroshock the chest and abdomen of the drone with a 220V voltage contact, so that the cuticle, mucus gland and glans penis of the drone are completely everted, then the electroejaculation is completed; Drone semen collection: After completing the electroshock operation on the drone, use a pipette to aspirate 100 μL of Kiev buffer solution, and flush the dark brown substance (i.e., semen) around the everted glans penis caused by electroshock, so that it flows into an EP tube along the semen collection channel. This semen collection method ensures the vitality and quantity of sperm, and significantly reduces the impurities in the extracted semen.

[0012] Furthermore, the Kiev buffer solution is prepared by mixing 3.60 g of trisodium citrate dihydrate, 0.32 g of sodium bicarbonate, 0.06 g of potassium chloride, and 0.50 g of glucose, and then making up the volume to 100 mL with double distilled water, with a pH of 8.0.

[0013] Furthermore, the semen of a single drone collected is subjected to cryogenic centrifugation at 2000 rpm for 1 min, and the sperm enrichment rate can reach about 90%, and the sperm vitality can reach about 86%.

[0014] Furthermore, if the same operation is performed on multiple drones, semen with high enrichment and high vitality can be obtained.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The drone sperm extraction device of the present invention is reasonably designed and has strong functionality. The detachable drone fixation device on the upper part of the device of the present invention facilitates the operator to take and place the drone and clean the device; the heightening of both sides of the semen collection channel effectively avoids semen overflow, and the structure with a narrow bottom and a wide top is conducive to semen flowing into the lower EP tube. The semen collection uses a disposable enzyme - free EP tube, the inner wall of which is extremely smooth, reducing liquid adsorption and residue, the tube wall is clear and easy to observe, and it has no DNA and RNA enzymes, effectively avoiding cross - contamination between semen, which is significantly better than the traditional use of slender capillary tubes for semen collection, which is prone to wall residue and blockage. Using the device for efficient extraction of semen from Apis cerana cerana drones of the present invention, semen without impurity contamination, high enrichment and high vitality can be obtained.

[0016] Moreover, the device is easy to operate, small and portable, and can be applied to bee breeding farms, bee research bases in universities, and household bee farmers, etc. On the one hand, for the artificial insemination of Chinese honeybee queens, the device effectively solves the problems of low efficiency in collecting sperm from drones, impurities in semen extraction, and low semen concentration. On the other hand, for drones with discovered excellent traits, if one wants to preserve the excellent germplasm, after expanding the reproduction of the excellent drones, the device can be used for batch collection and storage of semen, which is convenient for carrying out relevant research on the stored semen in the later stage.

[0017] 2. The method for extracting and collecting drone semen achieves high-efficiency and pollution-free sperm collection The method for collecting drone semen in the present invention greatly reduces the pollution of semen by impurities such as the mucus gland, and at the same time, the design of ten in a row for the three main components realizes the high-efficiency sperm collection of drones. In this method, sperm collection from drones is carried out in a super-clean workbench using a storage-type AC variable-frequency device. The chest and abdomen of the drone are electroshocked with a 220V voltage contact, so that the drone is subjected to appropriate electrical stimulation, causing the glans penis and the cutin to turn outwards instantaneously and hang above the semen collection channel, avoiding the semen from contacting the operator's fingers or the body surface of the drone. In addition, in this method, 100 mL of Kiev buffer solution is used to rinse the glans penis, so that the semen on the surface of the glans penis enters the sterile EP tube below together with the buffer solution to avoid sperm dehydration and death. Through experimental verification, the dosage of Kiev buffer solution in this method not only ensures that the semen can be completely rinsed, and the number of sperm in 1 mL of buffer solution reaches 7*10 8 . This method facilitates the use and storage of the collected semen and ensures the pollution-free of the semen, with strong usability.

[0018] 3. The cryogenic centrifugation enrichment method for drone semen provides high-vitality and high-density semen For the cryogenic centrifugation enrichment method of drone semen in the present invention, an EP tube containing a mixture of 100 mL of Kiev buffer solution and semen is processed in a 4°C cryogenic centrifuge at 2000 rpm for 1 minute. Through preliminary experimental verification, under the treatment of this rotation speed and time, the sperm enrichment rate in 100 mL of the mixed solution reaches about 90%, the sperm vitality is about 86%, and the number of sperm in 1 μL is 9*10 7 or so. The sperm vitality and enrichment rate are the best in the same batch of experimental groups. This method effectively avoids the waste of the extracted semen, and at the same time realizes the non-destructive enrichment of sperm, providing high-density and high-vitality sperm for queen artificial insemination. It solves the problem that the semen extracted from Chinese honeybee drones traditionally is less and viscous, and it is difficult for the number of sperm to reach the required amount for queen insemination.

[0019] The present invention effectively solves the difficulties in sperm collection from Chinese honeybee drones, and helps to promote the mature development of artificial insemination of Chinese honeybees. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the structural diagram of the sperm collection device for Chinese honeybee drones of the present invention; Figure 2 isFigure 1 Side view; Figure 3 is a flow chart of the method for highly efficient extraction and enrichment of semen of Apis cerana cerana drones in the present invention. Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. Components of the embodiments of the present invention generally described and illustrated in the figures herein can be arranged and designed in a variety of different configurations. Therefore, the detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the protection scope of the present invention.

[0021] It should be noted that like reference numerals and letters denote like items in the following figures, and thus, once an item is defined in one figure, it does not require further definition and explanation in subsequent figures. In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship in which the inventive product is usually placed during use. These are only for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus should not be construed as a limitation of the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive distinction and cannot be construed as indicating or implying relative importance. In addition, terms such as "horizontal" and "vertical" do not mean that the components are required to be absolutely horizontal or hanging, but can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined. In the description of the present invention, it should also be noted that unless otherwise clearly defined and limited, the terms "set", "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected, or indirectly connected through an intermediate medium, and can be the internal communication of two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations. Embodiment

[0022] I. A sperm collection device for Apis cerana cerana drones See Figure 1 and Figure 2 , a sperm extraction device for the drone of Apis cerana, comprising an extraction piston 1, a drone inserter 2 and a semen collection channel 3, and further comprising a sampling table 4, a movable semen collection table 5 and an EP tube 6. The extraction piston 1 is used to fix the drone in the drone inserter 2, so that the 2nd - 3rd tergites at the tail of the drone extend out of the opening of the drone inserter 2, facilitating semen collection; the drone inserter 2 is cylindrical and has a frustum shape (the diameter is larger at the top and smaller at the bottom), and a fan-shaped hollow with a length of 10 mm is provided near the lower opening, which is connected by two circular arcs with a width of 2 mm, used to prevent the drone from flying away and just being able to place an electric shock contact for sperm extraction operation. The drone inserter 2 is movably arranged on the semen collection channel 3 and forms a 45° angle, facilitating the electric shock sperm extraction operation; the drone inserter 2 is used to fix mature drones and cooperate with the extraction piston 1 to extract sperm from the drones. A semen collection channel 3 is provided below the drone inserter 2, and this channel is a pipe groove structure that is wider at the top and narrower at the bottom (the pipe groove is 10 mm long and about 2 mm deep), preventing semen overflow during collection; an EP tube 6 (1.5 mL) is arranged below the semen collection channel 3 to receive the semen washed in by Kiev buffer solution for subsequent enrichment and vitality determination of the semen.

[0023] The extraction piston 1, the drone inserter 2, the semen collection channel 3 and the corresponding EP tubes 6 are set on the movable semen collection table 5 in groups of 5 - 20, and the design method of one row or multiple rows realizes the efficient sperm extraction of drones.

[0024] The sperm extractor is used in conjunction with a storage type AC variable frequency device, and the voltage (current) generated by the storage type AC variable frequency device is used to give an electric shock to the drone for 1 - 2 s, prompting the drone to defecate while the penis turns outwards and secretes sperm, so as to efficiently obtain semen.

[0025] II. A method for efficient extraction and enrichment of semen from drones of Apis cerana See Figure 3 , the method of the present invention includes steps such as drone cultivation, drone fixation and electric shock sperm extraction, drone semen collection and drone semen enrichment, specifically as follows: S1: Drone cultivation S1.1: Drone preparation The first method is to perform CO2 anesthesia and induction of oviposition (anesthesia time 8 min, carbon dioxide concentration 75%) on the selected virgin queens with agile locomotor ability and good developmental status on the 5th and 6th days after emergence. After anesthesia, cut off 1 / 3 of the wing tips evenly to prevent them from going out for mating, and add an escape prevention sheet at the hive entrance, so that they only lay male eggs in the beehive.

[0026] The second method is to use a controller to control the egg-laying queen on the drone comb to force the queen to lay eggs.

[0027] After the queen bee lays eggs, two methods are used to regularly reward-feed the bee colony and timely supplement the brood combs, and the egg-laying and capping situations of the queen bee are observed.

[0028] S1.2: Drone marking 24 hours before the emergence of drones, place the drone brood combs in a clean indoor constant temperature and humidity incubator (temperature 33±2°C, humidity 53±2%). After the drones emerge, mark the drones of each age with different colors and then put them back into the original bee colony for rearing. Drones aged 16 - 22 days are selected for semen extraction during artificial insemination.

[0029] S2: Drone fixation and electroejaculation Select drones at the mature age (16 - 22 days), carefully place them into the drone fixator with their abdomens facing up and the 2 - 3 dorsal plates of the tails extending out of the fixing device, making the chest and abdomen of the drones face the electroejaculation port, and then install the extraction piston to stabilize the position of the drones; for semen extraction, use a storage type AC variable frequency device, and simultaneously electroejaculate the chest and abdomen of the drones with a 220V voltage contact, so that the cuticle, mucus gland and glans penis of the drones are completely everted, then the electroejaculation for semen extraction is completed.

[0030] S3: Drone semen collection After the electroejaculation operation of the drones is completed, use a pipette gun to aspirate 100 μL of Kiev buffer (3.60 g of sodium citrate, 0.32 g of sodium bicarbonate, 0.06 g of potassium chloride, 0.50 g of glucose are mixed and made up to 100 mL with double distilled water, pH 8.0), and flush the dark brown substance around the everted glans penis, which is the semen, so that it flows into the EP tube along the semen collection channel. This semen collection method ensures the viability and quantity of sperm, and significantly reduces the impurities in the extracted semen.

[0031] S4: Drone semen enrichment For drone semen extraction, electroejaculate a single sexually mature drone each time. After experimental verification, perform a freezing centrifugation treatment on the collected semen of a single drone at 2000 rpm for 1 min. The sperm enrichment rate can reach about 90% and the sperm viability can reach about 86%. Transfer 8 μL of the semen enriched at the bottom of the EP tube into another clean EP tube, and perform the same operation on multiple drones, then highly enriched and highly viable semen can be obtained.

[0032] The present invention provides a highly efficient drone semen extraction device to solve the problems of a large amount of impurities and low sperm viability during semen extraction. That is, in a laminar flow hood, use a storage type AC variable frequency device to electroejaculate the chest and abdomen of the drones with a 220V voltage contact, so that the drones are subjected to appropriate electrical stimulation to cause the glans penis and cuticle to evert instantaneously, and then collect the semen by flushing the glans penis with Kiev buffer, effectively avoiding semen contamination during extraction.

[0033] In addition, to address the phenomenon of insufficient utilization of semen during traditional semen collection, this method proposes to extract semen using an efficient semen collection device, in combination with specific centrifugation speeds and times of a refrigerated centrifuge, to fully enrich the collected clean semen without compromising sperm motility, meeting the requirements of saving high-quality drone bees and making full use of their semen.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than limiting them. Those of ordinary skill in the art should understand that any modifications or equivalent replacements made to the technical solutions of the present invention without departing from the purpose and scope of the present technical solution shall be covered within the scope of the claims of the present invention.

Claims

1. A sperm extraction device for Apis cerana cerana drones, characterized in that, It includes an extraction piston, a drone inserter, and a semen collection channel, and also includes a sampling table, a movable semen collection table, and an EP tube; the extraction piston is provided at the upper port of the drone inserter; the drone inserter is cylindrical and has a frustum shape. There are two fan-shaped hollow openings near the lower opening. The drone inserter is used to fix mature drones and cooperate with the extraction piston to extract semen from the drones; a semen collection channel is provided below the drone inserter, and an EP tube is arranged below the semen collection channel to collect the extracted semen.

2. The Apis cerana drone sperm extraction device according to claim 1, wherein The drone inserter is movably arranged on the semen collection table and is set at an angle of 45°, which is convenient for electroejaculation operation.

3. The Apis cerana cerana drone sperm extraction device according to claim 1, wherein The semen collection channel is a pipe groove structure that is wider at the top and narrower at the bottom to prevent semen spillage during collection.

4. The Apis cerana cerana drone sperm extraction device according to claim 1, characterized in that, The extraction piston, the drone inserter, the semen collection channel, and the corresponding EP tube arrays are arranged on the semen collection table of the sampling table, and are designed in one row or multiple rows.

5. The Apis cerana cerana drone sperm extraction device according to claim 4, characterized in that, The number of the arrays is 5 - 20 groups to achieve efficient semen extraction.

6. A method for efficient extraction and enrichment of semen from Apis cerana cerana drones, characterized in that, It includes steps such as drone cultivation, drone fixation and electroejaculation, drone semen collection, and drone semen enrichment; among them, Drone fixation and electroejaculation: Select mature-aged drones, place them in the drone fixator with their abdomens facing up and the 2 - 3 dorsal plates of the tails extending out of the fixing device, so that the chest and abdomen of the drones face the electroejaculation port, and then install the extraction piston to stabilize the position of the drones; For semen extraction, a storage type AC variable frequency device is used, and the chest and abdomen of the drones are simultaneously electroshocked with a 220V voltage contact head, so that the cuticle, mucus gland, and glans penis of the drones are completely everted, then the electroejaculation is completed; Drone semen collection: After the electroshock operation of the drones is completed, use a pipette gun to aspirate 100 μL of Kiev buffer solution, and flush the dark brown substance, that is, semen, around the everted glans penis caused by electroshock, so that it flows into the EP tube along the semen collection channel. This semen collection method ensures the viability and quantity of sperm, and significantly reduces the impurities in the extracted semen.

7. The method for highly efficient extraction and enrichment of semen of Apis cerana cerana drones according to claim 6, characterized in that, The Kiev buffer solution is composed of 3.60 g of trisodium citrate dihydrate, 0.32 g of sodium bicarbonate, 0.06 g of potassium chloride, and 0.50 g of glucose. After mixing, it is made up to 100 mL with double-distilled water, and the pH is 8.

0.

8. The method for highly efficient extraction and enrichment of semen of Apis cerana cerana drones according to claim 6, characterized in that, The semen of a single drone collected is subjected to cryogenic centrifugation at 2000 rpm for 1 min, and the sperm enrichment rate can reach about 90% and the sperm motility can reach about 86%.

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