Device and method for improving preservation time of porcine semen and sperm motility

Abstract

The present invention is applicable to the field related to pig reproduction, and provides a device and method for improving the preservation time of pig semen and sperm motility. The device for improving the preservation time of pig semen and sperm motility includes: a filtering cup, a primary microporous filtering membrane, a drainage bag, a precipitation cup, a secondary microporous filtering membrane module, a collection cup, and an adjustable negative pressure electric suction device. The bottom of the filtering cup is connected to the precipitation cup, and the secondary microporous filtering membrane module is arranged at the bottom of the precipitation cup. The drainage bag is sleeved on the cup mouth of the precipitation cup, and the primary microporous filtering membrane is arranged above the drainage bag. The precipitation cup is also provided with an air extraction port and a diversion port, wherein the air extraction port is communicated with the collection cup through a connecting pipe, and the diversion port is connected to the adjustable negative pressure electric suction device through a connecting pipe. The present invention greatly improves the motility of unqualified semen through a bionic environment + gradient collection + nano protective liquid.

Description

Technical Field

[0001] The present invention relates to the field related to pig reproduction, and specifically to a device and method for improving the preservation time of pig semen and sperm motility. Background Art

[0002] After pig semen is collected, it is generally filtered with filter paper, then the concentration is detected, diluted, and sub-packaged. Most of the filter paper materials are non-woven fabrics, and the micropore diameter is about 100 microns. Due to factors such as production process, material, and price, their quality varies greatly. Even multi-layer filtration cannot fully filter dead sperm and adherent sperm, and can only filter the larger-diameter colloids in the semen. In the production process of pig semen, colloids, dead sperm, and fragmented impurities inevitably exist. And sperm has the characteristic of anisotropism (also known as turbidity-seeking). Colloids, dead sperm, and fragmented impurities will attract surrounding live sperm to adhere and aggregate into clusters, getting more and more, resulting in a reduction in the number of effective sperm. At the same time, dead sperm produce a large amount of reactive oxygen species (ROS), which react with the sperm plasma membrane to cause lipid peroxidation of the sperm plasma membrane, resulting in damage to the sperm plasma membrane and a decrease in sperm motility. The above factors will lead to production problems such as shortened semen preservation time, low sperm motility, unsuitability for breeding, and increased cost of destroying unqualified semen. Another problem that cannot be ignored is that due to the lack of appropriate technical means, semen with unqualified motility in semen production is often directly discarded, resulting in a substantial increase in semen production costs and a huge waste of the genetic resources of high-value boars.

[0003] Due to the large ejaculate volume of pigs (150 - 500 mL / ejaculation) and the large single insemination volume (60 - 80 mL, about 1.5 - 3 billion effective sperm), the sperm swim-up method, Percoll density gradient centrifugation method, Pure Sperm centrifugation method, etc. commonly used in human reproductive centers in hospitals are not applicable to pig semen treatment. Currently, there is no good method to solve the above problems.

[0004] Therefore, in view of the above current situation, there is an urgent need to provide a device and method for improving the preservation time of pig semen and sperm motility to overcome the deficiencies in current practical applications. Summary of the Invention

[0005] The purpose of the present invention is to provide a device and method for improving the preservation time of pig semen and sperm motility, aiming to solve the problems in the above background art.

[0006] The present invention is implemented as follows. A device for improving the preservation time of pig semen and sperm motility includes: a filtering cup, a primary micro-porous filtering membrane, a drainage bag, a precipitation cup, a secondary micro-porous filtering membrane module, a collection cup, and an adjustable negative-pressure electric suction device. The bottom of the filtering cup is connected to the precipitation cup. The secondary micro-porous filtering membrane module is arranged at the bottom of the precipitation cup. The drainage bag is sleeved at the cup mouth of the precipitation cup, and the primary micro-porous filtering membrane is arranged above the drainage bag. An air extraction port and a diversion port are also arranged on the precipitation cup. The air extraction port is communicated with the collection cup through a connecting pipe, and the diversion port is connected to the adjustable negative-pressure electric suction device through a connecting pipe.

[0007] As a further solution of the present invention: The filtering cup is a round barrel made of borosilicate glass without a bottom. The diameter of the filtering cup is 10 - 15 cm. One end of the filtering cup is equipped with a fixing device, and an internal thread is arranged inside the fixing device. An external thread that cooperates with the internal thread is arranged on the precipitation cup.

[0008] As a further solution of the present invention: The primary micro-porous filtering membrane is a hydrophilic nylon mesh.

[0009] As a further solution of the present invention: The thickness of the primary micro-porous filtering membrane is 10 - 40 µm, and the diameter is the same as that of the filtering cup. The micropore diameter of the primary micro-porous filtering membrane is 40 - 60 µm, and the micropore spacing is 20 - 40 µm.

[0010] As a further solution of the present invention: The drainage bag is made of polyethylene with a thickness of 10 - 20 µm. The diameter of the drainage bag is the same as that of the precipitation cup, and the micropore spacing is 20 - 40 µm.

[0011] As a further solution of the present invention: The precipitation cup is a round cup made of borosilicate glass, and the diameter of the precipitation cup is the same as that of the filtering cup. The air extraction port is located on the side of the precipitation cup, 20 - 50 mm away from the cup mouth and protrudes. The diversion port is directly below the air extraction port, 10 - 20 mm away from the cup bottom and protrudes.

[0012] As a further solution of the present invention: The secondary micro-porous filtering membrane module includes a base and a secondary micro-porous filtering membrane. The base is made of polycarbonate, and the diameter of the base is slightly smaller than that of the precipitation cup. The secondary micro-porous filtering membrane is a hydrophilic nylon mesh.

[0013] As a further solution of the present invention: The thickness of the secondary micro-porous filtering membrane is 10 - 40 µm, the micropore diameter is 20 - 30 µm, and the diameter of the secondary micro-porous filtering membrane is the same as that of the precipitation cup, and the micropore spacing is 10 - 20 µm.

[0014] As a further solution of the present invention: The collection cup is a glass beaker, and the connecting pipe is a plastic pipe.

[0015] A method for improving the preservation time of porcine semen and sperm motility, using the device for improving the preservation time of porcine semen and sperm motility as described above. This method includes the following steps:

[0016] Step 1: Physiological regulation before semen collection, specifically including the following sub-steps:

[0017] 48 hours before semen collection, optimize the antioxidant capacity of boars by feeding a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast;

[0018] 2 hours before semen collection, place the boar in a bionic light environment to simulate the light conditions before natural mating and stimulate testosterone secretion;

[0019] After the physiological regulation before semen collection is completed, collect the raw semen of the boar;

[0020] Step 2: After collecting the raw semen of the boar, place the device in a bionic environment to simulate a water bath. The water bath temperature is 37 ± 0.5 °C, and dynamically adjust the water bath environment parameters through the temperature control module, including: the carbon dioxide concentration simulating the internal environment of the boar reproductive tract is 5.0 - 6.5%, the pH value is adjusted to 7.2 - 7.6, the intermittent low-frequency vibration is adjusted to a frequency of 1 - 3 Hz and an amplitude of 0.5 - 1 mm. At the same time, start the adjustable negative pressure electric suction device, and set the negative pressure value to -50 to -80 kPa, and dynamically adjust it according to the semen flow rate;

[0021] Step 3: Pour the fresh raw semen of the boar collected in Step 1 into the filter cup. Under the action of negative pressure and gravity, the colloid and impurities can be filtered through the primary microporous filter membrane;

[0022] The semen passing through the primary microporous filter membrane flows along the drainage bag to the secondary microporous filter membrane. The dead sperm and fragmented impurities in the semen precipitate to the bottom. The sperm with tail deformities and weak sperm cannot pass through the secondary microporous filter membrane due to their weak swimming ability. After the semen filtration is completed, let it stand and precipitate for 30 minutes;

[0023] Step 4: After standing, collect the sperm in stages and gradients through the diversion port, specifically:

[0024] S101: Collect the sperm with the strongest head activity that penetrates the secondary microporous filter membrane by negative pressure suction in the first 10 minutes;

[0025] S102: In the subsequent 20 minutes, use pulsed negative pressure, turn on / off at intervals of 5 seconds, to collect the remaining highly motile sperm, and introduce the screened sperm into a collection cup pre-filled with an arginine-trehalose composite protective solution with concentrations of 0.5 mM and 5 mM through a connecting tube to form a directional laminar interface between the sperm and the protective solution;

[0026] Step 6: Mix the semen in the collection cup with the nano-liposome diluent at a volume ratio of 1:4. The diluent contains the following components:

[0027] L101, basic diluent: 2.5 g / L of glucose, 1.2 g / L of sodium citrate, 0.1 g / L of ethylenediaminetetraacetic acid;

[0028] L102, functional additive: 0.1 μM of melatonin, 50 U / mL of superoxide dismutase (SOD), 0.05% of nano-α-tocopherol liposome;

[0029] Among them, the particle size of the nano-α-tocopherol liposome is 50 - 100 nm;

[0030] After mixing, aliquot into anti-freeze polyethylene bags and store using programmed cooling:

[0031] N101, the first stage: cool from 37°C to 4°C at a rate of -0.5°C / min;

[0032] N102, the second stage: cool from 4°C to -196°C at a rate of -5°C / min and store in liquid nitrogen for a long time.

[0033] Compared with the prior art, the beneficial effects of the present invention are:

[0034] 1. Through pre-semen collection physiological regulation (feed additive + bionic light), the sperm motility in the experimental group is increased to 93.7%, the malformation rate is reduced to 8.4%, and the testosterone level and antioxidant capacity are significantly enhanced;

[0035] 2. The present invention adopts a phased strategy of continuous negative pressure suction in the first 10 minutes and subsequent 2-minute pulsed negative pressure (opened / closed every 5 seconds), combined with the directional laminar interface of the arginine-trehalose composite protective solution. The enrichment rate of highly motile sperm reaches 92%, the residual amount of fragmented impurities is reduced to 3%, the sperm membrane integrity is increased to 94%, and at the same time, mechanical damage to sperm caused by direct semen drop is avoided;

[0036] 3. The present invention adds nano-α-tocopherol liposome to the diluent to target and repair lipid peroxidation damage of the sperm membrane. At the same time, 0.1 g / L of ethylenediaminetetraacetic acid (EDTA) is used to chelate metal ions to inhibit ROS generation. After storing for 168 h, the sperm motility still reaches 80%, and the plasma membrane integrity is increased by 18%;

[0037] 4. The present invention adopts two-stage programmed cooling to effectively reduce the physical damage of ice crystals to sperm. The semen storage time is extended to 192 h, and the passing rate of motility (≥70%) is increased by 25%;

[0038] 5. Through the synergistic effect of bionic environment treatment, gradient collection, and nano-protective liquid, the repair rate of semen with unqualified vitality (≤60%) is increased from 56% of the traditional method to 89%. The number of effective spermatozoa doubles, significantly reducing the waste of genetic resources. At the same time, it has the advantages of simple structure and is suitable for application in high-throughput pig semen production.

[0039] The present invention removes colloid, dead sperm, and fragmented impurities in semen through methods such as filtration, precipitation, and upstream, solving problems such as shortened semen preservation time and low sperm vitality. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0041] Figure 1 It is a schematic structural diagram of the present invention.

[0042] Figure 2 It is a schematic structural diagram of the filtration cup in the present invention.

[0043] Figure 3 It is a schematic structural diagram of the primary microporous filtration membrane in the present invention.

[0044] Figure 4 It is a schematic structural diagram of the drainage bag in the present invention.

[0045] Figure 5 It is a schematic structural diagram of the precipitation cup in the present invention.

[0046] Figure 6 It is a schematic structural diagram of the secondary microporous filtration membrane module in the present invention.

[0047] Figure 7 It is a schematic structural diagram of the collection cup in the present invention.

[0048] Figure 8 It is a schematic structural diagram of the adjustable negative pressure electric suction device in the present invention.

[0049] Figure 9 It is a sperm motility detection chart processed by the conventional method.

[0050] Figure 10 It is a sperm motility detection chart processed by the present invention.

[0051] Figure 11 It is a detection chart before processing unqualified semen.

[0052] Figure 12 It is a detection diagram after processing unqualified semen.

[0053] In the attached drawings: 1 - filter cup, 11 - internal thread, 2 - primary microporous filter membrane, 3 - drainage bag, 4 - precipitation cup, 41 - external thread, 42 - air extraction port, 43 - diversion port, 5 - secondary microporous filter membrane module, 51 - base, 52 - secondary microporous filter membrane, 6 - collection cup, 7 - adjustable negative pressure electric suction device. Specific implementation manners

[0054] Next, the technical solutions of the present invention will be clearly and completely described in conjunction with the attached drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0055] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the attached drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0056] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installation", "connection", "connection" 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 it 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 circumstances.

[0057] The following further explains and illustrates the present invention in combination with specific implementation manners.

[0058] Please refer to Figures 1 - 12, A device for improving the preservation time of pig semen and sperm motility provided by an embodiment of the present invention includes a filtering cup 1, a primary microporous filtering membrane 2, a drainage bag 3, a precipitation cup 4, a secondary microporous filtering membrane module 5, a collection cup 6, and an adjustable negative pressure electric suction device 7. The bottom of the filtering cup 1 is connected to the precipitation cup 4. The secondary microporous filtering membrane module 5 is arranged at the bottom of the precipitation cup 4. The drainage bag 3 is sleeved on the mouth of the precipitation cup 4. The primary microporous filtering membrane 2 is arranged above the drainage bag 3. An air extraction port 42 and a diversion port 43 are also arranged on the precipitation cup 4. Among them, the air extraction port 42 is communicated with the collection cup 6 through a connecting pipe, and the diversion port 43 is connected to the adjustable negative pressure electric suction device 7 through a connecting pipe;

[0059] The filtering cup 1 is a round barrel made of borosilicate glass without a bottom. The diameter of the filtering cup 1 is 10 - 15 cm. One end of the filtering cup 1 is equipped with a fixing device. The fixing device is made of ordinary plastic material, and an internal thread 11 is arranged inside the fixing device. An external thread 41 that cooperates with the internal thread 11 is arranged on the precipitation cup 4;

[0060] The primary microporous filtering membrane 2 is a hydrophilic nylon mesh;

[0061] The thickness of the primary microporous filtering membrane 2 is 10 - 40 µm, and its diameter is the same as that of the filtering cup 1. The micropore diameter of the primary microporous filtering membrane 2 is 40 - 60 µm, and the micropore spacing is 20 - 40 µm;

[0062] The drainage bag 3 is made of polyethylene material with a thickness of 10 - 20 µm. The diameter of the drainage bag 3 is the same as that of the precipitation cup 4, and the micropore spacing is 20 - 40 µm;

[0063] The precipitation cup 4 is a round cup made of borosilicate glass, and the diameter of the precipitation cup 4 is the same as that of the filtering cup 1. The air extraction port 42 is located on the side of the precipitation cup 4 and is 20 - 50 mm away from the cup mouth and protrudes, which is convenient for connecting the adjustable negative pressure electric suction device 7 through a connecting pipe with a valve; The diversion port 43 is directly below the air extraction port 42 and is 10 - 20 mm away from the cup bottom and protrudes, which is convenient for connecting the collection cup 6 through a connecting pipe with a valve;

[0064] The secondary microporous filtering membrane module 5 includes a base 51 and a secondary microporous filtering membrane 52. The base 51 is made of polycarbonate material, and the diameter of the base 51 is slightly smaller than that of the precipitation cup 4. The secondary microporous filtering membrane 52 is a hydrophilic nylon mesh;

[0065] The thickness of the secondary microporous filtering membrane 52 is 10 - 40 µm, the micropore diameter is 20 - 30 µm, and the diameter of the secondary microporous filtering membrane 52 is the same as that of the precipitation cup 4. The micropore spacing is 10 - 20 µm. When in use, the base 51 is placed in the precipitation cup 4, and the secondary microporous filtering membrane 52 is covered on the base 51;

[0066] The collection cup 6 is a commercially available ordinary glass beaker, the connecting tube is a commercially available ordinary plastic tube, and the adjustable negative pressure electric suction device 7 is a commercially available ordinary model.

[0067] In an embodiment of the present invention, when using this device, the device is placed in a water bath pot with a water bath temperature of 37 °C. The adjustable negative pressure electric suction device 7 is started, and the freshly collected raw boar semen is poured into the filtration cup 1. Under the action of negative pressure and gravity, the colloid and impurities can be filtered through the primary microporous filtration membrane 2; the semen passing through the primary microporous filtration membrane 2 flows along the drainage bag 3 to the secondary microporous filtration membrane 52 of the secondary microporous filtration membrane module 5, avoiding mechanical damage to sperm caused by direct semen dropping; the dead sperm and fragmented impurities in the semen precipitate to the bottom, and the sperm with tail deformities and weak sperm cannot pass through the secondary microporous filtration membrane 52 due to their weak swimming ability. After the semen filtration is completed, it is allowed to precipitate for 30 minutes and then introduced into the collection cup 6 through the connecting tube. The sperm with strong swimming ability after screening are in the collection cup 6, and their viability and preservation time are greatly improved. The semen is diluted and preserved according to the conventional method; at the same time, it has the advantages of simple structure and being suitable for application in high-throughput pig semen production.

[0068] The present invention removes colloid, dead sperm, and fragmented impurities in semen through methods such as filtration, precipitation, and upstream, solving problems such as shortened semen preservation time and low sperm viability.

[0069] Please refer to Figures 1 - 12 , a method for improving the preservation time of pig semen and sperm viability provided by an embodiment of the present invention, uses the above-mentioned device for improving the preservation time of pig semen and sperm viability. This method includes the following steps:

[0070] Step 1: Physiological regulation before semen collection, specifically including the following sub-steps:

[0071] 48 hours before semen collection, by feeding a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast, the antioxidant capacity of boars is optimized;

[0072] 2 hours before semen collection, the boar is placed in a bionic light environment to simulate the light conditions before natural mating and stimulate testosterone secretion;

[0073] After the physiological regulation before semen collection is completed, the raw boar semen is collected;

[0074] Step 2: After the raw boar semen is collected, the device is placed in a bionic environment to simulate a water bath pot with a water bath temperature of 37 ± 0.5 °C, and the water bath environment parameters are dynamically adjusted through a temperature control module, including:

[0075] The carbon dioxide concentration simulating the internal environment of the boar reproductive tract is 5.0% - 6.5%, the pH value is adjusted to 7.2 - 7.6, the intermittent low-frequency vibration is adjusted to a frequency of 1 - 3 Hz and an amplitude of 0.5 - 1 mm. At the same time, start the adjustable negative pressure electric suction device 7, and set the negative pressure value to -50 to -80 kPa, and dynamically adjust it according to the semen flow rate;

[0076] Step 3: Pour the fresh raw boar semen collected in Step 1 into the filter cup 1. Under the action of negative pressure and gravity, the semen can filter out colloid and impurities through the primary microporous filter membrane 2;

[0077] Step 4: The semen passing through the primary microporous filter membrane 2 flows along the drainage bag 3 to the secondary microporous filter membrane 52. The dead sperm and fragmented impurities in the semen precipitate to the bottom. The sperm with tail deformities and weak sperm cannot pass through the secondary microporous filter membrane 52 due to their weak swimming ability. After the semen filtration is completed, let it stand and precipitate for 30 min;

[0078] Step 5: After standing, collect sperm in stages through the diversion port 43. Specifically:

[0079] S501: Collect the sperm with the strongest head activity that penetrates the secondary microporous filter membrane 52 by negative pressure suction in the first 10 min;

[0080] S502: In the subsequent 20 min, use pulsed negative pressure, turn on / off at intervals of 5 seconds, to collect the remaining highly viable sperm, and introduce the screened sperm into the collection cup 6 pre-filled with arginine-trehalose composite protective solution with concentrations of 0.5 mM and 5 mM respectively through the connecting tube to form a directional laminar interface between the sperm and the protective solution;

[0081] Step 6: Mix the semen in the collection cup 6 with the nanoliposome diluent at a volume ratio of 1:4. The diluent contains the following components:

[0082] L101: Basic diluent: glucose 2.5 g / L, sodium citrate 1.2 g / L, ethylenediaminetetraacetic acid 0.1 g / L;

[0083] L102: Functional additive: melatonin 0.1 μM, superoxide dismutase (SOD) 50 U / mL, nanoscale α-tocopherol liposome 0.05%;

[0084] Among them, the particle size of the nanoscale α-tocopherol liposome is 50 - 100 nm;

[0085] After mixing, sub-pack it into anti-freeze polyethylene bags and store it by programmed cooling:

[0086] N101: The first stage: cool from 37 °C to 4 °C at -0.5 °C / min;

[0087] N102, Second stage: Cool from 4°C to -196°C at a rate of -5°C / min and store in liquid nitrogen for a long time.

[0088] The sperm detection experiment is as follows:

[0089] I. Materials and Methods

[0090] 1. Materials and Methods

[0091] 1.1. Test Population

[0092] The test population comes from the core boars in the core herd of a Canadian line core breeding farm in Zhangzhou City, Fujian Province. All boars are raised in open pens with cement slatted floors in the same pig house, fed regularly and quantitatively every day, and given free access to water.

[0093] 1.2. Experimental Instruments

[0094] Table 1-1 Names and Manufacturers of Main Experimental Instruments and Equipment

[0095]

[0096] 1.3. Experimental Reagents

[0097] The ultrapure water used in the experiment is self-made, and the semen diluent powder is provided by Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.

[0098] 2. Experimental Contents and Methods

[0099] 2.1. Experimental Contents

[0100] 2.1.1. Sperm Motility Experiment: Divide the semen of the boars that passed the preliminary test equally, and detect the sperm motility after conventional dilution (control group) and conventional dilution after treatment with the new method (experimental group) respectively, and compare the effects of the two treatment methods on sperm motility.

[0101] 2.1.2. Sperm Preservation Time Experiment: Directly dilute the semen of the boars that passed the preliminary test conventionally (control group) and conventionally dilute it after treatment with the new method (experimental group), then put it into an incubator at 17°C, turn it over twice a day, and detect the sperm motility at 48h, 72h, 96h, 120h, 144h, 168h, and 192h.

[0102] 2.1.3. Experiment on the Treatment of Unqualified Semen: Dilute the semen of the boars that failed the preliminary test conventionally after treatment with the new method (experimental group), and then detect the sperm motility to observe the effect of the new treatment method on improving sperm motility.

[0103] 2.2. Experimental Methods

[0104] 2.2.1. Collection of Porcine Semen

[0105] Collect fresh semen by the fist-grip method at the boar station and send it to the laboratory within 10 minutes.

[0106] 2.2.2. Semen biological detection experiment using the CASA sperm quality analysis system

[0107] Use the CASA sperm quality analysis system for analysis. The automatic image analysis results provided by the CASA sperm quality analysis system are objective, highly accurate, have good repeatability, and can quickly process analysis data. It can provide an objective quantitative analysis of sperm samples and is currently widely used in large-scale pig semen production.

[0108] a. Turn on the microscope and the supporting computer, preheat the microscope hot stage, and prepare conventional commercial semen, ordinary glass slides, cover glasses, Leja 4-chamber sperm detection slides, 20 μL pipette tips, pipettes, etc.;

[0109] b. Aspirate the semen and add it to the chamber of a disposable sperm counting chamber slide. Use the CASA system to intercept 3 fields of view to observe sperm motility and status, and save and export the detection results as EXCEL.

[0110] 2.3. Data processing

[0111] Use EXCEL 2019 software to organize the original data and R V4.3.2 software for data analysis.

[0112] 2.3.1. Randomly divide 20 healthy adult boars into two groups:

[0113] Control group: Feed conventional feed 48 hours before semen collection, and the semen collection environment is ordinary white light;

[0114] Experimental group: Feed a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast 48 hours before semen collection, and place them in a biomimetic light environment 2 hours before semen collection;

[0115] Table 2-1 Comparison of physiological regulation before pre-semen collection

[0116]

[0117] As can be seen from Table 2-1, L-arginine promotes the synthesis of nitric oxide (NO) and improves testicular microcirculation. Selenium yeast, as a cofactor of glutathione peroxidase, directly scavenges reactive oxygen species.

[0118] The SOD activity in the experimental group increased by 64.4%, indicating that the combination of the two significantly enhanced the antioxidant ability of sperm and reduced lipid peroxidation damage. In the biomimetic light environment, the photoreceptor cells of the boar retina were activated, and testosterone secretion was stimulated through the hypothalamus-pituitary-gonadal axis. The testosterone concentration in the experimental group increased by 71.2%, directly promoting spermatogenesis and maturation and reducing the malformation rate.

[0119] Initial sperm motility (%): Immediately detected by the CASA system after semen collection;

[0120] Sperm malformation rate (%): Evaluated by the Diff-Quik staining method;

[0121] Testosterone concentration (ng / mL): Blood was collected 1 h before semen collection and determined by ELISA;

[0122] Antioxidant index (SOD activity, U / mg protein): Superoxide dismutase activity in semen.

[0123] 2.3.2. Compare the two collection methods and conduct result statistics

[0124] Table 2-2 Effects of optimized bionic environment parameters on sperm motility

[0125]

[0126] As can be seen from Table 2-2, the complete bionic environment (Group 4) is significantly better than the single-parameter improvement (Groups 2 and 3), proving that the synergistic effect of CO2, pH, and vibration can improve sperm motility and preservation stability. Compared with Group 1, the motility increased by 7% after treatment, and the motility was still 6% higher than that of the control group after 48 h of preservation, reflecting the creative value of the bionic environment.

[0127] 2.3.3. Compare the two collection methods and conduct result statistics

[0128] Table 2-3 Effects of staged gradient collection on the enrichment rate of highly motile sperm

[0129]

[0130] As can be seen from Table 2-3, staged gradient collection (Method B) reduces mechanical damage through pulsed negative pressure, the enrichment rate of highly motile sperm increases by 14%, and the impurity residue is reduced to 3%, which is significantly better than Method A. Combined with the directional laminar interface of the arginine-trehalose protective solution, the sperm membrane integrity increases by 12%.

[0131] 2.3.4. Divide the semen of the same batch into 3 groups for preservation and detect the results after 168 h of preservation

[0132] Table 2-4 Comparison of cryopreservation effects between nano-liposome diluent and traditional diluent

[0133]

[0134] As can be seen from Table 2-4, the nano-liposome diluent (Group 3) significantly inhibits lipid peroxidation (a 66% reduction) by targeting the delivery of α-tocopherol, and both the viability and acrosome integrity rate far exceed those of the control group. Combined with programmed cooling (two-stage cooling), the viability still reaches 80% after 168 hours of preservation, which is 25% higher than that of Group 1 (64%), demonstrating the effectiveness of the diluent in the solution of the present invention.

[0135] 2.3.5. Select substandard semen with a viability ≤ 60% and treat it with traditional methods and the method of the present invention respectively

[0136] Table 2-5 Comparison of the repair effects of substandard semen

[0137]

[0138] As can be seen from Table 2-5, the solution of the present invention, through a biomimetic environment + gradient collection + nano-protecting solution, increases the viability of substandard semen to 89%, doubles the number of effective sperm, and reduces the malformation rate to 6% (usually requiring a viability ≥ 70%).

[0139] 2.3.6. Statistics of the experimental results of sperm viability

[0140] Table 2-6 Statistics of the experimental results of sperm viability

[0141]

[0142] After the same semen is processed, the semen viability details in the experimental group significantly exceed those of the control group, with a significant difference.

[0143] 2.3.7. Statistics of the experimental results of sperm preservation time

[0144] Table 2-7 Statistics of the experimental results of sperm preservation time

[0145]

[0146] According to the general requirements of the pig-raising industry, the sperm viability before insemination should not be lower than 70%. The sperm preservation reagent in the experimental group is significantly extended, exceeding the control group by 48 hours, with a significant difference.

[0147] 2.3.8. Experiment on the treatment of substandard semen

[0148] Table 2-8 Statistics of the treatment results of substandard semen

[0149]

[0150] After the semen with unqualified viability is processed, the sperm viability is significantly improved. See Figure 11 、 Figure 12 。

[0151] II. Experimental results

[0152] The experimental results show that the device and method described in this application have significant effects on improving sperm motility and sperm preservation time, and can also meet the qualified standards after treating unqualified semen.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the preservation time of porcine semen and sperm motility, characterized in that, It is carried out by using a device for improving the preservation time of boar semen and sperm motility. The method includes the following steps: Step 1: Physiological regulation before semen collection, specifically including the following sub-steps: 48 hours before semen collection, optimize the antioxidant capacity of boars by feeding a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast; 2 hours before semen collection, place the boar in a bionic light environment to simulate the light conditions before natural mating and stimulate testosterone secretion; After the physiological regulation before semen collection is completed, collect the raw semen of the boar; Step 2: After collecting the raw semen of the boar, place the device in a bionic environment to simulate a water bath. The water bath temperature is 37±0.5°C, and dynamically adjust the water bath environment parameters through the temperature control module, including: the carbon dioxide concentration simulating the internal environment of the boar reproductive tract is 5.0%-6.5%, the pH value is adjusted to 7.2-7.6, the intermittent low-frequency vibration is adjusted to a frequency of 1-3 Hz and an amplitude of 0.5-1 mm. At the same time, start the adjustable negative pressure electric suction device (7), and set the negative pressure value to -50 to -80 kPa, and dynamically adjust it according to the semen flow rate; Step 3: Pour the fresh raw semen of the boar collected in Step 1 into the filter cup (1). Under the action of negative pressure and gravity, the colloid and impurities can be filtered through the primary microporous filter membrane (2); The semen passing through the primary microporous filter membrane (2) flows along the drainage bag (3) to the secondary microporous filter membrane (52). The dead sperm and fragmented impurities in the semen precipitate to the bottom. The sperm with tail deformities and weak sperm cannot pass through the secondary microporous filter membrane (52) due to their weak swimming ability. After the semen filtration is completed, let it stand and precipitate for 30 minutes; Step 5: After standing, collect sperm in stages and gradients through the diversion port (43), specifically: S501: Collect the sperm with the strongest head activity penetrating the secondary microporous filter membrane (52) by negative pressure suction in the first 10 minutes; S502: In the subsequent 20 minutes, use pulsed negative pressure, turn on / off at intervals of 5 seconds, to collect the remaining highly motile sperm, and introduce the screened sperm into the collection cup (6) pre-filled with arginine-trehalose composite protective solution with concentrations of 0.5 mM and 5 mM respectively through the connecting pipe to form a directional laminar interface between the sperm and the protective solution; Step 6: Mix the semen in the collection cup (6) with the nanoliposome diluent at a volume ratio of 1:

4. The diluent contains the following components: L101: Basic diluent: 2.5 g / L of glucose, 1.2 g / L of sodium citrate, 0.1 g / L of ethylenediaminetetraacetic acid; L102: Functional additive: 0.1 μM of melatonin, 50 U / mL of superoxide dismutase (SOD), 0.05% of nano-α-tocopherol liposome; Among them, the particle size of the nano-α-tocopherol liposome is 50-100 nm; After mixing, sub-pack it into anti-freeze polyethylene bags and store it by programmed cooling: N101: The first stage: Cool from 37°C to 4°C at a rate of -0.5°C / min; N102: The second stage: Cool from 4°C to -196°C at a rate of -5°C / min and store it in liquid nitrogen for a long time; Among them, the device for improving the preservation time of porcine semen and sperm motility includes a filtering cup (1), a primary microporous filtering membrane (2), a drainage bag (3), a precipitation cup (4), a secondary microporous filtering membrane module (5), a collection cup (6), and an adjustable negative pressure electric suction device (7). The bottom of the filtering cup (1) is connected to the precipitation cup (4). The secondary microporous filtering membrane module (5) is arranged at the bottom of the precipitation cup (4). The drainage bag (3) is sleeved on the cup mouth of the precipitation cup (4). The primary microporous filtering membrane (2) is arranged above the drainage bag (3). An air extraction port (42) and a diversion port (43) are also arranged on the precipitation cup (4). Among them, the air extraction port (42) is communicated with the collection cup (6) through a connecting pipe, and the diversion port (43) is connected to the adjustable negative pressure electric suction device (7) through a connecting pipe.

2. The method for improving the preservation time of porcine semen and sperm motility according to claim 1, characterized in that The filtering cup (1) is a round barrel made of borosilicate glass without a bottom. The diameter of the filtering cup (1) is 10 - 15 cm. One end of the filtering cup (1) is equipped with a fixing device, and an internal thread (11) is arranged inside the fixing device. An external thread (41) that cooperates with the internal thread (11) is arranged on the precipitation cup (4).

3. The method for improving the preservation time of porcine semen and sperm motility according to claim 2, characterized in that, The thickness of the primary microporous filtering membrane (2) is 10 - 40 µm, and its diameter is the same as that of the filtering cup (1). The micropore diameter of the primary microporous filtering membrane (2) is 40 - 60 µm, and the micropore spacing is 20 - 40 µm.

4. A method for improving the preservation time of porcine semen and sperm motility according to claim 1, characterized in that, The drainage bag (3) is made of polyethylene with a thickness of 10 - 20 µm. The diameter of the drainage bag (3) is the same as that of the precipitation cup (4), and the micropore spacing is 20 - 40 µm.

5. A method for improving the preservation time of porcine semen and sperm motility according to claim 1, characterized in that, The precipitation cup (4) is a round cup made of borosilicate glass, and the diameter of the precipitation cup (4) is the same as that of the filtering cup (1). The air extraction port (42) is located on the side of the precipitation cup (4) and is 20 - 50 mm away from the cup mouth and protrudes. The diversion port (43) is directly below the air extraction port (42) and is 10 - 20 mm away from the cup bottom and protrudes.

6. A method for improving the preservation time of porcine semen and sperm motility according to claim 1, characterized in that The secondary microporous filtering membrane module (5) includes a base (51) and a secondary microporous filtering membrane (52). The base (51) is made of polycarbonate, and the diameter of the base (51) is slightly smaller than that of the precipitation cup (4). The secondary microporous filtering membrane (52) is a hydrophilic nylon mesh.

7. A method for improving the preservation time of porcine semen and sperm motility according to claim 6, characterized in that, The thickness of the secondary microporous filtering membrane (52) is 10 - 40 µm, the micropore diameter is 20 - 30 µm, and the diameter of the secondary microporous filtering membrane (52) is the same as that of the precipitation cup (4), and the micropore spacing is 10 - 20 µm.

8. A method for improving the preservation time of porcine semen and sperm motility according to claim 1, characterized in that, The collection cup (6) is a glass beaker, and the connecting pipe is a plastic pipe.

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