Artificial insemination method for improving breeding efficiency of scirpus juncoides

Abstract

The invention discloses an artificial insemination method for improving the breeding efficiency of Juncus spp.. The method comprises the following steps: S1, collecting seminal fluid and screening qualified seminal fluid; s2, diluting, activating and storing the semen; s3, preparing female insects; s4, trace directional intracapsular injection is carried out; s5, postoperative management and fertilization detection, wherein eggs are collected, and the fertilization rate and the hatching rate are evaluated. According to the method, four qualified screening standards for the window-juncus semen are put forward for the first time, and low-quality semen is effectively prevented from entering an insemination link; the constructed diluent has a good preservation effect on the seminal fluid, provides higher flexibility for the use of the seminal fluid, and remarkably improves the utilization rate of the seminal fluid; according to the integrated scheme of semen screening, dilution activation, low-temperature preservation and short catheter accurate injection, capsule inlet trace directional infusion is provided for the genital cavity structure of the female leucopus juncoides, the insemination rate of 65%-75% and the hatching rate of 60%-65% can be stably obtained, and the stability requirements of artificial breeding and experimental breeding are met.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of artificial propagation of insects, and more particularly, to an artificial insemination method for improving the propagation efficiency of Pyrocoelia, so as to improve the efficiency of single male mating and the egg production of female insects. BACKGROUND

[0002] Pyrocoelia spp. belongs to the class of insects, the order of Coleoptera, and the family of Lampyridae, and is an important terrestrial firefly. The eggs, larvae and adults of Pyrocoelia spp. can emit light, and have the functions of ecological indication, scientific research and cultural appreciation, and are widely used in ecological research, rare population conservation, popular science display and ornamental breeding.

[0003] At present, the propagation of Pyrocoelia spp. completely depends on natural mating, and the natural mating has problems such as low utilization rate of male insects, long mating time and unstable fertilization rate. At present, there is no report on artificial insemination technology for any variety of firefly, and Pyrocoelia spp. has no sperm packet in anatomical characteristics. The key to artificial insemination of insects lies in maintaining the activity and movement performance of sperm. The existing diluent is mostly based on ordinary insect culture medium, but lacks an energy supply system, resulting in rapid inactivation of sperm under low-temperature preservation. Therefore, there is an urgent need for a technical system that can screen high-quality sperm, improve utilization rate, maintain sperm activity, and cooperate with microinjection optimized for the structure of the female reproductive tract of Pyrocoelia spp. to improve the fertilization rate. In view of this, the present application provides an artificial insemination method for improving the propagation efficiency of Pyrocoelia spp. SUMMARY

[0004] The present application aims to provide an artificial insemination method for improving the propagation efficiency of Pyrocoelia spp.

[0005] According to one aspect of the present application, an artificial insemination method for improving the propagation efficiency of Pyrocoelia spp. is provided, comprising the following steps: S1. Sperm collection and qualified screening: collecting sperm of male insects, detecting the sperm activity rate, average curve movement speed VCL, straight line movement rate STR and concentration, and determining that the collected sperm of male insects is qualified sperm only when the activity rate is ≥80%, VCL is ≥120 μm / s, STR is ≥60%, and the concentration is ≥1×10 6 / μL; S2. Sperm dilution activation and preservation: diluting the qualified sperm with a diluent at a volume ratio of 1:1 to 2:1 to obtain diluted sperm, standing for 5 min at 25±1℃ to achieve the purpose of activating sperm, and then directly injecting the diluted sperm or storing the diluted sperm in cold storage for standby, wherein the diluent contains Grace insect culture medium, ATP-Na2, MgCl2, HEPES and penicillin-streptomycin; S3. Female preparation: selecting female insects that have been hatched for ≤24 h and have not yet flashed for courtship, fixing the female insects with the ventral surface upward, and exposing the female reproductive opening by lightly pressing with a blunt forceps; S4. Micro-directed intracystic injection: Under a stereomicroscope, a micro-quantitative injection system equipped with a micro-injection needle is used. The tip of the micro-injection needle is inserted 1.6-2.4 mm along the axis of the reproductive cavity-short duct lumen at an angle of 30°-45° relative to the female body axis. The slight resistance generated at the entrance of the short duct is used as a positioning signal to determine the injection termination position. Diluted semen is injected quantitatively at this termination position. After injection, the needle tip is retained for 2-4 seconds to reduce backflow, and then withdrawn along the insertion path. S5. Postoperative management and fertilization test: Place the female worms that have completed intracystic injection at 25±1℃ and 70-80% relative humidity, collect the eggs and evaluate the fertilization rate and hatching rate.

[0006] In some embodiments, the diluent uses Grace insect culture medium (1×) as the base solution and contains ATP-Na2 1-3 mmol / L, MgCl2 3-7 mmol / L, HEPES 8-12 mmol / L (pH = 7.2), penicillin 80-120 IU / mL, streptomycin 80-120 IU / mL, and the final osmotic pressure of the diluent is 320-340 mOsm / kg.

[0007] In some embodiments, the diluted semen in step S2 is refrigerated at 4±1°C for no more than 12 hours and then warmed to 25°C before being used for subsequent injection.

[0008] In some embodiments, the micro-quantitative injection system is a picoliter injection pump with a propulsion structure or a micro-syringe with a mechanical limit adjustment structure, the propulsion structure being able to control the flow rate for intracapsular quantitative infusion of diluted semen.

[0009] In some embodiments, the micro-injection needle is drawn from a capillary glass tube with a tip diameter of 60-80 μm and an inner diameter of 25-35 μm, and its tip is ground to form a 28°-32° single-sided bevel structure.

[0010] In some implementations, the tip of the micro-injection needle is ground to form a 30° single-sided bevel structure.

[0011] In some embodiments, in step S4, the tip of the micro-injection needle is inserted along the axis of the reproductive cavity-short duct cavity at an insertion angle of 30°-45° relative to the female body axis.

[0012] In some embodiments, in step S4, 0.15-0.25 μL of diluted semen is injected at the termination position at a rate of 0.02-0.05 μL / s.

[0013] In some implementations, a single male insect can fertilize 8-15 female insects, and the fertilization rate of the female insects after fertilization is ≥65%, and the hatching rate is ≥60%.

[0014] According to another aspect of the present invention, an artificial insemination method for improving the reproductive efficiency of *Fireflyia pulcherrima* is provided, which is applied to obtaining *Fireflyia pulcherrima* fertilized eggs, larvae, or adults, and in the conservation, commercial breeding, and larval supply system construction of *Fireflyia pulcherrima* artificial populations.

[0015] The beneficial effects of the present invention are: (1) The present invention proposes for the first time four qualified screening criteria for fenestrated semen (viability ≥80%, VCL ≥120 μm / s, STR ≥60%, concentration ≥1×10 6 (strains / μL), this screening mechanism effectively prevents low-quality semen from entering the insemination process; (2) The diluent constructed in this invention contains an ATP-Mg energy system. After the semen is diluted, it can be either allowed to stand and activated before injection, or it can be stored at 4±1℃ for no more than 12 hours. During the storage process, the sperm motility decreases by less than 15%. This invention has a good preservation effect on diluted semen. The dual-mode mechanism of dilution and storage provides greater flexibility for the use of semen, meets the needs of batch insemination and time mismatch operations, and significantly improves the semen utilization rate. (3) This invention proposes a “micro-directed infusion at the sac entrance” for the first time for the reproductive cavity structure of female firefly through an integrated solution of “sperm screening-dilution activation-low temperature preservation-precise injection via short catheter”. This achieves the technical goal of controllable infusion of highly active semen and output of high fertilization rate. A single male can fertilize 8-15 females, which improves the utilization rate and fertilization efficiency of firefly semen. It provides a standardized and controllable technical route for the artificial breeding of firefly and has strong potential for industrial application. (4) The method of the present invention can stably obtain a fertilization rate of 65-75% and a hatching rate of 60-65% in actual operation, which meets the stability requirements of artificial breeding and experimental reproduction, and is about 30% higher than natural mating. Furthermore, the method has low equipment requirements and the operation steps can be standardized, making it suitable for scientific ecological research, conservation of rare window firefly populations, live display for ornamental tourism, and the construction of commercial artificial breeding systems. Attached Figure Description

[0016] Figure 1 This is a framework diagram of an artificial insemination method for improving the reproductive efficiency of fireflies according to the present invention.

[0017] Figure 2 This is a comparison chart of the motility of diluted and undiluted semen in Example 2 of the present invention. Detailed Implementation

[0018] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0019] Source of male and female insects: In October 2022, pupated individuals of *Fireflyia pulcherrima* were collected from rice paddies in the suburbs of Fang County, Hubei Province. 248 female insects and 100 male insects that had emerged ≤24 h and had not shown courtship flashes were selected. Individuals with abdominal damage or hardened elytra were removed and used in the following examples.

[0020] Example 1

[0021] An artificial insemination method to improve the reproductive efficiency of fireflies, such as... Figure 1 As shown, it includes the following steps: S1. Semen Collection and Screening: (a) Semen collection: Male free semen was collected using the intercourse interruption method and immediately stored at 25°C for later use. (b) Parameter detection: 5 μL of semen was placed on a hemocytometer and continuously photographed for 0.5 s (60 fps) with a 40× objective lens on a 25℃ constant temperature microscopic imaging platform. The TrackMate module of ImageJ software was used to automatically track ≥30 sperm motility trajectories. After excluding stationary particles, the viability (%), VCL (μm / s), STR (%) and concentration (sperm / μL) were automatically output. (c) Judgment criteria: when the survival rate is ≥80%, VCL is ≥120μm / s, STR is ≥60%, and the concentration is ≥1×10 6 A semen sample is considered qualified if it has a density of 100 μL / μL. All four indicators must be met simultaneously. Samples that do not meet the standards will not be allowed to proceed to the insemination process.

[0022] S2. Semen dilution, activation, and preservation: (a) Preparation of dilution solution: Grace insect medium (1×) 1000 mL, ATP-Na2 2.00 mmol / L, MgCl2 5.00 mmol / L, HEPES 10.0 mmol / L (adjust pH to 7.20 with appropriate amount of NaOH), penicillin 100 IU / mL, streptomycin 100 IU / mL, final osmotic pressure adjusted to 330 mOsm / kg; (b) Dilute the qualified semen collected in step S1 with the diluent at a volume ratio of 1:1 to obtain diluted semen. Let it stand at 25±1℃ for 5 min to activate the semen. The diluted semen can be injected directly or stored at 4±1℃ for no more than 12 h. Before use, it should be warmed to 25℃.

[0023] S3. Preparation of female insects: Select female insects that have emerged ≤24 h and have not shown signs of courtship. After mildly anesthetizing the female insects to be fertilized with CO2 for 2-3 minutes, fix them on the wax plate with their ventral side facing up. Use blunt forceps to gently press the terminal abdominal segment to fully expose the genital cavity opening and keep it open.

[0024] S4. Micro-directed intracapsular injection: (a) Injection system configuration: A picoliter injection pump with a propulsion structure or a microsyringe with a mechanical limit adjustment structure is used as the micro-quantitative injection system. This micro-quantitative injection system can control the flow rate for intracapsular quantitative infusion of diluted semen. A capillary glass micro-injection needle is selected. The micro-injection needle is 25-30 mm in total length, with an insertion section of 5-6 mm, an outer diameter of 30-80 μm at the tip, and an inner diameter of 25-35 μm. The needle tip is mechanically ground to form a 30° single-sided bevel structure to reduce tissue tearing and resistance fluctuations when entering the short catheter. The micro-injection needle is installed at the top of the micro-quantitative injection system. (b) Directional Insertion Path and Resistance Positioning: Under a stereomicroscope, the tip of the micro-injection needle is inserted along the axis of the reproductive cavity-short duct, simulating the insertion direction of the male's mating organ, at an angle of 30°-45° relative to the female's body axis, with an insertion depth controlled at 1.6-2.4 mm. A slight resistance is felt when the needle tip touches the entrance of the short duct; this resistance serves as a positioning signal to determine the injection termination point, preventing the needle tip from penetrating the seminal vesicle and ensuring that semen is concentrated in the vesicle entrance area. During the procedure, the injection depth is controlled at approximately 1.6-2.4 mm, a range that is essentially consistent with the effective insertion range of the male during natural mating, covering the area from the female's reproductive cavity to the seminal vesicle opening, thus achieving precise semen delivery. (c) Micro-volume constant-rate injection control: After positioning, 0.2 μL of diluted semen is injected at a constant rate of 0.05 μL / s using an injection pump with a constant-rate propulsion structure or a micro-syringe with a limiting structure. The needle is left in place for 3 seconds and then withdrawn along the original path to reduce semen backflow. In this method, the volume of diluted semen injected into a single female insect is controlled at 0.15-0.25 μL, and the sperm count is no less than 7.5 × 10⁻⁶. 4 The injection volume is sufficient to meet the sperm count required for fertilization, while avoiding excessive pressure on the reproductive cavity or semen backflow due to excessive volume.

[0025] S5. Postoperative management and fertilization testing: After anesthesia is relieved, a single female insect is placed in an incubation box (25±1℃, RH 80%), and eggs are collected within 6-48 hours. The eggshell hardening-sinking method is used for rapid determination: eggs that sink to the bottom are fertilized eggs, and those that float are unfertilized eggs. The fertilization rate is calculated. The hatching rate is obtained by continuing incubation for 10-50 days.

[0026] Example 2: Practical Operation Verification of the Method

[0027] In this embodiment, semen was collected from 25 male insects collected in Fang County, Hubei Province in October 2022, following the method in Example 1, and artificial insemination was performed on 108 female insects. After artificial insemination, eggs were collected and incubated, and the fertilization rate and hatching rate were calculated.

[0028] The results showed that 25 samples of male semen were collected and tested in this embodiment, of which 5 samples did not meet the four screening criteria set in step S1 of Example 1 (viability ≥80%, VCL ≥120 μm / s, STR ≥60%, concentration ≥1×10⁻⁶). 6 (Sperm / μL) were excluded, resulting in a semen qualification rate of 80%. A single qualified male insect can be collected 2-3 times, and the amount of semen or sperm obtained each time will still meet the requirements for fertilization by the female insect.

[0029] like Figure 2 As shown, the preferred initial sperm motility was 91.3% ± 2.1%. After treatment with the diluent from step S2 in Example 1, and storage at 4 ± 1°C for 12 hours, the sperm motility remained at 84.0% ± 2.7%, with a decrease of only 7.3% (<15%) within 12 hours. Furthermore, VCL and STR remained within a stable range, indicating continued effective fertilization capability. This demonstrates that the diluent and storage conditions of the present invention extend the semen storage time to 12 hours.

[0030] Semen that had not been treated with the diluent was stored at 4±1℃ for 12 hours, and the viability decreased by 34.3% (>30%), indicating that the diluent of the present invention has a significant protective effect on the viability of semen.

[0031] In this embodiment, 108 females were successfully fertilized, with a fertilization rate of 71.2% ± 4.8% (n=102) and a hatching rate of 65.2% ± 5.1%. Egg development was stable, and no obviously deformed individuals were produced. This demonstrates that the method of the present invention provides a standardized and controllable technical route for the artificial breeding of *Fireflyia pulcherrima*, effectively improving fertilization efficiency by approximately 30% compared to natural mating.

[0032] Example 3: Validation experiment of artificial insemination without semen screening and diluent treatment

[0033] In this embodiment, 15 male insects and 60 female insects from the same batch were selected. Semen from the male insects was collected, but the semen was not screened for quality. Grace medium was used instead of the diluent in Example 1 to dilute the collected semen 1:1. Artificial insemination was performed on the 60 female insects according to steps S2-S5 in Example 1, and the fertilization rate and hatching rate were calculated.

[0034] The results showed that 39 female insects were injected in this embodiment (the micro-injection needle blockage rate was 35%), with an fertilization rate of 52.4% ± 7.1% and a hatching rate of 46.0% ± 6.4%. Compared with Example 2, the fertilization success rate in this embodiment was significantly lower, and the needle blockage rate was significantly increased, indicating that sperm screening and diluent treatment were not performed, resulting in a lower artificial insemination rate and hatching rate. It is evident that the diluent of this invention can maintain sperm motility, ensure good needle patency, effectively delay sperm motility decline during storage, and improve low-temperature preservation stability.

[0035] Example 4: Controlled Experiment on Natural Mating of Fireflies

[0036] Twenty-five males and fifty females from the same batch were selected and allowed to mate freely for two nights in a mating cage under natural photoperiod. The ambient temperature was maintained at 25±1℃ and humidity at 70-80%. The fertilization rate was 43.3%±6.1%, and the hatching rate was 38.7%±5.9%, significantly lower than the artificial insemination results of Example 2 of this invention. This demonstrates that the artificial insemination method of this invention, through an integrated approach of "sperm screening-dilution activation-low-temperature preservation-precise injection via short catheter," and the "micro-directed infusion at the sac entrance" proposed for the reproductive cavity structure of the female *Fireflyia pulcherrima*, achieves the technical goals of controllable infusion of highly active semen and high fertilization rate output. In actual operation, a stable fertilization rate of 65-75% and a hatching rate of 60-65% can be obtained, which is about 30% higher than natural mating, meeting the stability requirements for artificial breeding and experimental reproduction.

[0037] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An artificial insemination method for improving the reproductive efficiency of fireflies, characterized in that, Includes the following steps: S1. Semen Collection and Screening: Free semen was collected from male sperm larvae, and sperm motility, mean velocities (VCL), stroma (STR), and sperm concentration were measured. Only sperm with motility ≥80%, VCL ≥120 μm / s, STR ≥60%, and concentration ≥1×10⁻⁶ were eligible for screening. 6 When the sperm count is 1 / μL, the collected male semen is considered to be qualified semen; S2. Semen dilution, activation and preservation: The above qualified semen is diluted with the diluent at a volume ratio of 1:1 to 2:1 to obtain diluted semen. The diluted semen is then allowed to stand at 25±1℃ for 5 min to activate it. The diluted semen is then injected directly or stored under cold storage for later use. The diluent contains Grace insect culture medium, ATP-Na2, MgCl2, HEPES and penicillin-streptomycin. S3. Preparation of female insects: Select female insects that have emerged for ≤24 hours and have not shown flashing mating lights, fix them with their ventral side facing up, and gently press with blunt-tipped tweezers to expose the female insect's genital opening. S4. Micro-directed intracystic injection: Under a stereomicroscope, using a micro-quantitative injection system equipped with a micro-injection needle, the tip of the micro-injection needle is inserted 1.6-2.4 mm along the axis of the reproductive cavity-short duct lumen at an angle of 30°-45° relative to the female body axis. The slight resistance generated at the entrance of the short duct is used as a positioning signal to determine the injection termination position. The diluted semen is quantitatively injected at this termination position. After the injection, the needle tip is retained for 2-4 seconds to reduce backflow, and then withdrawn along the insertion path. S5. Postoperative management and fertilization test: Place the female worms that have completed intracystic injection at 25±1℃ and 70-80% relative humidity, collect the eggs and evaluate the fertilization rate and hatching rate.

2. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, The diluent uses Grace insect medium (1×) as the base solution and contains ATP-Na2 1-3 mmol / L, MgCl2 3-7 mmol / L, HEPES 8-12 mmol / L (pH = 7.2), penicillin 80-120 IU / mL, streptomycin 80-120 IU / mL, and the final osmotic pressure of the diluent is 320-340 mOsm / kg.

3. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, In step S2, the diluted semen is refrigerated at 4±1℃ for no more than 12 hours and then warmed to 25℃ before being used for subsequent injection.

4. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, The micro-quantitative injection system is a picoliter injection pump with a propulsion structure or a micro-syringe with a mechanical limiting and adjusting structure. The propulsion structure can control the flow rate for intracapsular quantitative infusion of diluted semen.

5. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, The micro-injection needle is drawn from a capillary glass tube, with a tip diameter of 60-80μm and an inner diameter of 25-35μm. Its tip is ground to form a 28°-32° single-sided bevel structure.

6. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 5, characterized in that, The tip of the micro-injection needle is ground to form a 30° single-sided bevel structure.

7. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, In step S4, the tip of the micro-injection needle is inserted along the axis of the reproductive cavity-short duct cavity at an insertion angle of 30°-45° relative to the female worm's body axis.

8. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, In step S4, 0.15-0.25 μL of diluted semen is injected at the termination position at a rate of 0.02-0.05 μL / s.

9. The artificial insemination method for improving the reproductive efficiency of fireflies according to claim 1, characterized in that, A single male insect can fertilize 8-15 female insects, and the fertilization rate of the female insects after fertilization is ≥65%, and the hatching rate is ≥60%.

10. The artificial insemination method for improving the reproductive efficiency of *Fireflyia pulcherrima* as described in any one of claims 1 to 9 is used in obtaining fertilized eggs, larvae, or adults of *Fireflyia pulcherrima*, and in the conservation, commercial breeding, and larval supply system construction of artificial populations of *Fireflyia pulcherrima*.

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