Device and method for prolonging preservation time of boar semen and improving sperm motility

Abstract

The invention is suitable for the related field of pig breeding, and provides a device and method for prolonging the preservation time of pig semen and improving the sperm motility, and the device for prolonging the preservation time of the pig semen and improving the sperm motility comprises a filter cup, a primary microporous filter membrane, a drainage bag, a precipitation cup, a secondary microporous filter membrane module, a collection cup and an adjustable negative pressure electric aspirator, the bottom of the filter cup is connected with the precipitation cup, the secondary microporous filter membrane module is arranged at the bottom of the precipitation cup, the drainage bag is sleeved with a cup opening of the precipitation cup, the primary microporous filter membrane is arranged above the drainage bag, the precipitation cup is further provided with an air extraction opening and a flow guide opening, the air extraction opening is communicated with the collection cup through a connecting pipe, and the flow guide opening is communicated with the collection cup through a connecting pipe. According to the invention, through the bionic environment, gradient collection and nano protection liquid, the vitality of unqualified seminal fluid is greatly improved.

Description

Technical Field

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

[0002] After the pig semen is collected, it is usually filtered with filter paper to detect the concentration, dilute, and package. Most of the filter paper is made of non-woven fabric with a micropore diameter of about 100 microns. Due to factors such as production process, material, and price, its quality varies. Even multi-layer filtration cannot fully filter dead sperm and adherent sperm, and can only filter colloids with larger diameters in semen. In the process of pig semen production, due to the inevitable existence of colloid, dead sperm, and fragmented impurities, and sperm has anisotropy (also known as turbidity), colloid, dead sperm, and fragmented impurities will attract surrounding live sperm to adhere and aggregate into clusters, and the more they gather, the less effective sperm count will be. At the same time, dead sperm produce a large amount of reactive oxygen species (ROS) that react with the sperm plasma membrane to undergo lipid peroxidation, causing damage to the sperm plasma membrane and causing a decrease in sperm motility. The above factors will lead to production problems such as shortened semen storage time, low sperm motility and unsuitability for breeding, and increased costs for destroying unqualified semen. Another issue that cannot be ignored is that due to the lack of appropriate technical means, semen that does not meet the vitality requirements during semen production is often directly discarded, resulting in a significant increase in semen production costs and a huge waste of high-value boar genetic resources.

[0003] Due to the huge amount of pig ejaculation (150-500mL / time) and the large amount of single insemination (60-80mL, about 1.5-3 billion effective sperms), the sperm swim-up method, Percoll density gradient centrifugation method, Pure Sperm centrifugation method, etc., which are commonly used in the human reproductive center of the hospital, are not suitable for pig semen processing. There is currently no better way to solve the above problems.

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

[0005] The object of the present invention is to provide a device and method for improving the storage time and sperm motility of pig semen, aiming to solve the problems in the above-mentioned background technology.

[0006] The present invention is achieved in this way. A device for improving the preservation time and sperm motility of pig semen includes: a filter cup, a primary microporous filter membrane, a drainage bag, a sedimentation cup, a secondary microporous filter membrane module, a collection cup and an adjustable negative pressure electric aspirator. The bottom of the filter cup is connected to the sedimentation cup, the secondary microporous filter membrane module is arranged at the bottom of the sedimentation cup, the drainage bag sleeve is at the cup mouth of the sedimentation cup, the primary microporous filter membrane is arranged above the drainage bag, and the sedimentation cup is also provided with an air suction port and a diversion port, wherein the air suction port is connected to the collection cup through a connecting pipe, and the diversion port is connected to the adjustable negative pressure electric aspirator through a connecting pipe.

[0007] As a further solution of the present invention: the filter cup is a borosilicate glass barrel without a bottom, the diameter of the filter cup is 10-15 cm, a fixing device is installed at one end of the filter cup, and an internal thread is arranged in the fixing device, and the sedimentation cup is provided with an external thread used in conjunction with the internal thread.

[0008] As a further solution of the present invention: the primary microporous filtration membrane is a hydrophilic nylon mesh membrane.

[0009] As a further solution of the present invention: the thickness of the primary microporous filtration membrane is 10-40µm, the diameter is the same as the filter cup, the micropore diameter of the primary microporous filtration membrane is 40-60µm, and the micropore spacing is 20-40μm.

[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 sedimentation cup, and the micropore spacing is 20-40 μm.

[0011] As a further solution of the present invention: the sedimentation cup is a round cup made of borosilicate glass, and the diameter of the sedimentation cup is consistent with the diameter of the filter cup; the air suction port is located on the side of the sedimentation cup, 20-50 mm away from the cup mouth, and is protruding; the guide port is located directly below the air suction port, 10-20 mm away from the bottom of the cup, and is protruding.

[0012] As a further solution of the present invention: the secondary microporous filtration membrane module includes a base and a secondary microporous filtration membrane, the base is made of polycarbonate, and the diameter of the base is slightly smaller than the sedimentation cup; the secondary microporous filtration membrane is a hydrophilic nylon mesh membrane.

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

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

[0015] A method for improving the storage time and sperm motility of pig semen, using the above-mentioned device for improving the storage time and sperm motility of pig semen, the method comprises the following steps: Step 1: Pre-semen collection physiological regulation, including the following sub-steps: Optimize the boars’ antioxidant capacity by feeding them a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast 48 hours before semen collection; 2 hours before semen collection, the boars were placed in a bionic lighting environment to simulate the lighting conditions before natural mating and stimulate testosterone secretion; After the physiological regulation of pre-semen collection is completed, the boar semen is collected; Step 2: After the boar semen is collected, the device is placed in a bionic environment simulation water bath pot with a water bath temperature of 37±0.5℃, and the water bath environment parameters are dynamically adjusted through the temperature control module, including: the carbon dioxide concentration simulating the environment in 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 1-3Hz, an amplitude of 0.5-1mm, and the adjustable negative pressure electric aspirator is started at the same time, the negative pressure value is set to -50 to -80kPa, and is dynamically adjusted with the semen flow rate; Step 3: Pour the fresh boar semen collected in step 1 into the filter cup. Under the action of negative pressure and gravity, the semen can filter out colloid and impurities through the primary microporous filter membrane; Step 4: The semen that has passed through the primary microporous filter membrane flows along the drainage bag to the secondary microporous filter membrane. Dead sperm and fragmented impurities in the semen settle to the bottom. Sperm with deformed tails 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 settle for 30 minutes. Step 5: After the standing time is over, sperm is collected in stages through the diversion port, specifically: S101, in the first 10 minutes, the sperm with the strongest head activity that penetrated the secondary microporous filtration membrane was collected by negative pressure suction; S102, for the next 20 minutes, pulsed negative pressure was used, with an interval of 5 seconds to open / close, to collect the remaining high-motility sperm, and the selected sperm were introduced into a collection cup pre-filled with arginine-trehalose composite protective solution with concentrations of 0.5 mM and 5 mM through a connecting tube to form a directional laminar flow interface between the sperm and the protective solution; Step 6: Mix the semen in the collection cup with the nanoliposome diluent at a volume ratio of 1:4, wherein the diluent comprises the following components: L101, basic diluent: glucose 2.5g / L, sodium citrate 1.2g / L, EDTA 0.1g / L; L102, functional additives: melatonin 0.1µM, superoxide dismutase (SOD) 50U / mL, nano-sized α-tocopherol liposome 0.05%; Among them, the particle size of nano-sized α-tocopherol liposomes is 50-100nm; After mixing, pack into antifreeze polyethylene bags and store at programmed temperature: N101, first stage: from 37°C to 4°C at -0.5°C / min; N102, second stage: reduce the temperature from 4℃ to -196℃ at -5℃ / min, and store in liquid nitrogen for a long time.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Through pre-semen collection physiological regulation (feed additives + bionic lighting), the sperm motility of the experimental group increased to 93.7%, the deformity rate dropped to 8.4%, and the testosterone level and antioxidant capacity were significantly enhanced; 2. The present invention adopts a phased strategy of continuous negative pressure suction for the first 10 minutes and pulsed negative pressure for the subsequent 2 minutes (on / off at an interval of 5 seconds), combined with the directional laminar flow interface of the arginine-trehalose composite protective solution, the high-motility sperm enrichment rate reaches 92%, the residual amount of fragmented impurities is reduced to 3%, and the sperm membrane integrity is improved to 94%, while preventing the direct fall of semen from causing mechanical damage to the sperm; 3. The present invention uses nano-sized α-tocopherol liposomes added to the diluent to target and repair lipid peroxidation damage of sperm membrane, and at the same time, 0.1g / L ethylenediaminetetraacetic acid (EDTA) is used to chelate metal ions and inhibit ROS generation. After 168 hours of storage, sperm motility still reaches 80%, and plasma membrane integrity is improved by 18%; 4. The present invention adopts two-stage programmed cooling to effectively reduce the physical damage of ice crystals to sperm, extend the semen storage time to 192h, and increase the vitality rate (≥70%) by 25%; 5. The present invention improves the repair rate of semen with unqualified vitality (≤60%) from 56% of the traditional method to 89% through the synergistic effect of bionic environment treatment, gradient collection and nano-protective liquid, doubles the number of effective sperm, and significantly reduces 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.

[0017] The present invention removes colloid, dead sperm and fragmented impurities in semen by filtering, sedimentation and upstreaming, thereby solving the problems of shortened semen storage time and low sperm motility. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is a schematic diagram of the structure of the present invention.

[0020] Figure 2 It is a schematic diagram of the structure of the filter cup in the present invention.

[0021] Figure 3 It is a schematic diagram of the structure of the primary microporous filtration membrane in the present invention.

[0022] Figure 4 It is a schematic diagram of the structure of the drainage bag in the present invention.

[0023] Figure 5 It is a schematic diagram of the structure of the sedimentation cup in the present invention.

[0024] Figure 6 It is a schematic structural diagram of the secondary microporous filtration membrane module in the present invention.

[0025] Figure 7 It is a schematic diagram of the structure of the collecting cup in the present invention.

[0026] Figure 8 It is a schematic diagram of the structure of the adjustable negative pressure electric suction device in the present invention.

[0027] Fig. 9 This is a sperm motility test chart processed by conventional methods.

[0028] Fig.10 This is a sperm motility detection diagram processed by the present invention.

[0029] Fig.11 This is a picture of the inspection before processing of unqualified semen.

[0030] Fig.12 This is the test picture after processing of unqualified semen.

[0031] In the attached drawings: 1-filter cup, 11-internal thread, 2-primary microporous filtration membrane, 3-drainage bag, 4-sedimentation cup, 41-external thread, 42-vacuum port, 43-flow diversion port, 5-secondary microporous filtration membrane module, 51-base, 52-secondary microporous filtration membrane, 6-collection cup, 7-adjustable negative pressure electric aspirator. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are 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 cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0034] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "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 a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention will be understood according to specific circumstances.

[0035] The present invention is further explained below in conjunction with specific implementation modes.

[0036] See also Figure 1-Figure 12 , a device for improving the storage time and sperm motility of pig semen provided by an embodiment of the present invention comprises a filter cup 1, a primary microporous filter membrane 2, a drainage bag 3, a sedimentation cup 4, a secondary microporous filter membrane module 5, a collection cup 6 and an adjustable negative pressure electric aspirator 7, wherein the bottom of the filter cup 1 is connected to the sedimentation cup 4, the secondary microporous filter membrane module 5 is arranged at the bottom of the sedimentation cup 4, the drainage bag 3 is sleeved with the cup mouth of the sedimentation cup 4, the primary microporous filter membrane 2 is arranged above the drainage bag 3, and the sedimentation cup 4 is further provided with an air suction port 42 and a diversion port 43, wherein the air suction port 42 is connected to the collection cup 6 through a connecting pipe, and the diversion port 43 is connected to the adjustable negative pressure electric aspirator 7 through a connecting pipe; The filter cup 1 is a borosilicate glass barrel without a bottom. The diameter of the filter cup 1 is 10-15 cm. A fixing device is installed at one end of the filter cup 1. The fixing device is made of ordinary plastic material and has an internal thread 11. The sedimentation cup 4 is provided with an external thread 41 used in conjunction with the internal thread 11. The primary microporous filtration membrane 2 is a hydrophilic nylon mesh membrane; The thickness of the primary microporous filtration membrane 2 is 10-40 μm, and the diameter is the same as that of the filter cup 1. The micropore diameter of the primary microporous filtration membrane 2 is 40-60 μm, and the micropore spacing is 20-40 μm; 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 sedimentation cup 4, and the micropore spacing is 20-40 μm. The precipitation cup 4 is a round cup made of borosilicate glass, and the diameter of the precipitation cup 4 is consistent with the diameter of the filter cup 1; the air suction port 42 is located on the side of the precipitation cup 4, 20-50 mm away from the cup mouth, and is protrudingly arranged to facilitate the connection of the connecting pipe with a valve to the adjustable negative pressure electric suction device 7; the guide port 43 is located directly below the air suction port 42, 10-20 mm away from the bottom of the cup, and is protrudingly arranged to facilitate the connection of the collection cup 6 through the connecting pipe with a valve; The secondary microporous filtration membrane module 5 includes a base 51 and a secondary microporous filtration membrane 52. The base 51 is made of polycarbonate, and the diameter of the base 51 is slightly smaller than the sedimentation cup 4. The secondary microporous filtration membrane 52 is a hydrophilic nylon mesh membrane. The secondary microporous filtration membrane 52 has a thickness of 10-40 μm and a micropore diameter of 20-30 μm. The diameter of the secondary microporous filtration membrane 52 is the same as that of the precipitation cup 4, and the micropore spacing is 10-20 μm. When in use, the base 51 is placed in the precipitation cup 4, and the secondary microporous filtration membrane 52 is covered on the base 51; The collecting cup 6 is a common glass beaker available on the market, the connecting tube is a common plastic tube available on the market, and the adjustable negative pressure electric suction device 7 is a common model available on the market.

[0037] In an embodiment of the present invention, when the device is used, 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 collected fresh boar semen is poured 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; the semen passing through the primary microporous filter membrane 2 flows along the drainage bag 3 to the secondary microporous filter membrane 52 of the secondary microporous filter membrane module 5 to prevent the semen from falling directly and causing mechanical damage to the sperm; dead sperm and fragmented impurities in the semen are precipitated to the bottom, and sperm with deformed tails and weak sperm cannot pass through the secondary microporous filter membrane 52 due to their weak swimming ability. After the semen is filtered, it is precipitated for 30 minutes and introduced into the collection cup 6 through the connecting tube. The collection cup 6 contains sperm with strong swimming ability after screening, and its vitality and storage time are greatly improved. The semen is diluted and stored according to the conventional method; at the same time, it has the advantages of simple structure and suitable for application in high-throughput pig semen production; The present invention removes colloid, dead sperm and fragmented impurities in semen by filtering, sedimentation and upstreaming, thereby solving the problems of shortened semen storage time and low sperm motility.

[0038] See also Figure 1-Figure 12 The embodiment of the present invention provides a method for improving the storage time and sperm motility of pig semen, using the above-mentioned device for improving the storage time and sperm motility of pig semen, and the method comprises the following steps: Step 1: Pre-semen collection physiological regulation, including the following sub-steps: Optimize the boars’ antioxidant capacity by feeding them a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast 48 hours before semen collection; 2 hours before semen collection, the boars were placed in a bionic lighting environment to simulate the lighting conditions before natural mating and stimulate testosterone secretion; After the physiological regulation of pre-semen collection is completed, the boar semen is collected; Step 2: After the boar semen is collected, the device is placed in a bionic environment simulated water bath with a water bath temperature of 37±0.5℃. The water bath environment parameters are dynamically adjusted through the temperature control module, including: The carbon dioxide concentration of the simulated boar reproductive tract environment 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, and the adjustable negative pressure electric suction device 7 is started at the same time, and the negative pressure value is set to -50 to -80 kPa and is dynamically adjusted according to the semen flow rate; Step 3: Pour the fresh 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; Step 4: The semen that has passed through the primary microporous filtration membrane 2 flows along the drainage bag 3 to the secondary microporous filtration membrane 52, and the dead sperm and fragmented impurities in the semen settle to the bottom. The sperm with deformed tails 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 settle for 30 minutes; Step 5: After the standing time is over, sperm is collected in stages through the diversion port 43, specifically: S501, in the first 10 minutes, the sperm with the strongest head activity that penetrates the secondary microporous filter membrane 52 is collected by negative pressure suction; S502, for the next 20 minutes, pulsed negative pressure is used, which is turned on / off at intervals of 5 seconds to collect the remaining high-motility sperm, and the selected sperm are introduced into the collection cup 6 pre-filled with arginine-trehalose composite protective solution with concentrations of 0.5mM and 5mM respectively through the connecting tube to form a directional laminar flow 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, wherein the diluent comprises the following components: L101, basic diluent: glucose 2.5g / L, sodium citrate 1.2g / L, EDTA 0.1g / L; L102, functional additives: melatonin 0.1µM, superoxide dismutase (SOD) 50U / mL, nano-sized α-tocopherol liposome 0.05%; Among them, the particle size of nano-sized α-tocopherol liposomes is 50-100nm; After mixing, pack into antifreeze polyethylene bags and store at programmed temperature: N101, first stage: from 37°C to 4°C at -0.5°C / min; N102, second stage: reduce the temperature from 4℃ to -196℃ at -5℃ / min, and store in liquid nitrogen for a long time.

[0039] The sperm testing test is as follows: 1. Materials and Methods 1. Materials and methods 1.1 Experimental population The experimental population was from the core group of boars from a Canadian core breeding farm in Zhangzhou City, Fujian Province. All boars were raised in open pens on cement slatted floors in the same pig house, fed at regular times and quantitatively every day, and had free access to drinking water.

[0040] 1.2 Experimental instruments Table 1-1 Names and manufacturers of main experimental instruments and equipment

[0041] 1.3 Experimental Reagents The ultrapure water used in the experiment was homemade, and the semen dilution powder was provided by Jiangsu Zhongmu Beikang Pharmaceutical Co., Ltd.

[0042] 2. Experimental content and methods 2.1 Experimental content 2.1.1. Sperm motility experiment: The semen of the boars that passed the preliminary test was divided into equal parts, and the sperm motility was tested after conventional dilution (control group) and conventional dilution after treatment with the new method (experimental group), and the effects of the two treatment methods on sperm motility were compared.

[0043] 2.1.2. Sperm preservation time experiment: The boar semen that passed the preliminary test was directly diluted conventionally (control group) and the semen that was treated with the new method was diluted conventionally (experimental group) and then placed in a 17℃ constant temperature box, turned over twice a day, and the sperm motility was tested at 48h, 72h, 96h, 120h, 144h, 168h, and 192h.

[0044] 2.1.3. Experiment on treatment of unqualified semen: The semen of boars that initially failed the test was treated according to the new method and then diluted routinely (experimental group) and tested for sperm motility to observe the effect of the new treatment method on improving sperm motility.

[0045] 2.2 Experimental Methods 2.2.1. Collection of pig semen Fresh semen was collected by fist-grip method in boar stud and delivered to the laboratory within 10 min.

[0046] 2.2.2. CASA sperm quality analysis system semen biological detection experiment The CASA sperm quality analysis system is used for analysis. The automatic image analysis results provided by the CASA sperm quality analysis system have high objective accuracy, good repeatability, and rapid analysis data processing. It can provide an objective quantitative analysis of sperm samples and is currently widely used in large-scale pig semen production.

[0047] a. Turn on the microscope and the supporting computer, preheat the microscope hot stage, and prepare regular commercial semen, ordinary slides, cover slips, Leja 4-chamber sperm detection slides, 20uL pipette tips, pipettes, etc.; b. Aspirate semen and add it to the chamber of the disposable sperm counting pool slide. Use the CASA system to capture 3 fields of view to observe sperm motility and status. Save the test results and export them to EXCEL.

[0048] 2.3 Data Processing EXCEL 2019 software was used to organize the raw data, and R V4.3.2 software was used for data analysis.

[0049] 2.3.1. Randomly divide 20 healthy adult boars into two groups: Control group: The mice were fed with regular feed 48 h before semen collection, and the semen collection environment was normal white light; Experimental group: The mice were fed with a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast 48 h before semen collection and placed in a bionic lighting environment 2 h before semen collection; Table 2-1 Comparison of physiological regulation of pre-semen collection

[0050] As shown in Table 2-1, L-arginine improves testicular microcirculation by promoting the synthesis of nitric oxide (NO), and selenium yeast, as a cofactor of glutathione peroxidase, directly removes reactive oxygen species.

[0051] The SOD activity of the experimental group increased by 64.4%, indicating that the combination of the two significantly enhanced the antioxidant capacity of sperm and reduced lipid peroxidation damage. Under the bionic lighting environment, the boar retinal photoreceptor cells were activated, and the testosterone secretion was stimulated through the hypothalamus-pituitary-gonad axis. The testosterone concentration in the experimental group increased by 71.2%, which directly promoted spermatogenesis and maturation and reduced the deformity rate.

[0052] Initial sperm motility (%): tested by CASA system immediately after semen collection; Sperm deformity rate (%): evaluated by Diff-Quik staining method; Testosterone concentration (ng / mL): Blood was collected 1 h before semen collection and measured by ELISA; Antioxidant index (SOD activity, U / mg protein): Superoxide dismutase activity in semen.

[0053] 2.3.2. Compare the two collection methods and make statistical results Table 2-2 Effect of optimization of bionic environment parameters on sperm motility

[0054] 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 storage stability. Compared with Group 1, the vitality increased by 7% after treatment, and after 48 hours of storage, the vitality was still 6% higher than that of the control group, reflecting the creative value of the bionic environment.

[0055] 2.3.3. Compare the two collection methods and make statistical results Table 2-3 Effect of phased gradient collection on the enrichment rate of high-motility sperm

[0056] As can be seen from Table 2-3, the staged gradient collection (method B) reduces mechanical damage through pulsed negative pressure, increases the enrichment rate of high-motility sperm by 14%, and reduces the residual impurities to 3%, which is significantly better than method A. Combined with the directional laminar flow interface of arginine-trehalose protective solution, the sperm membrane integrity is improved by 12%.

[0057] 2.3.4. Semen from the same batch was divided into three groups for storage and the test results were obtained after 168 hours of storage. Table 2-4 Comparison of cryopreservation effects between nanoliposome diluent and traditional diluent

[0058] As shown in Tables 2-4, the nanoliposome diluent (Group 3) significantly inhibited lipid peroxidation (reduced by 66%) by targeted delivery of α-tocopherol, and the activity and acrosome integrity rate were far higher than those of the control group. Combined with programmed cooling (two-stage cooling), the activity was still 80% after 168 hours of storage, an increase of 25% over Group 1 (64%), reflecting the effectiveness of the diluent in the scheme of the present invention.

[0059] 2.3.5. Select unqualified semen with vitality ≤ 60% and treat them using the traditional method and the method of the present invention respectively Table 2-5 Comparison of repair effects of unqualified semen

[0060] It can be seen from Tables 2-5 that the scheme of the present invention, through bionic environment + gradient collection + nano-protective liquid, can increase the motility of unqualified semen to 89%, double the number of effective sperm, and reduce the deformity rate to 6% (usually requiring motility ≥ 70%).

[0061] 2.3.6. Statistics of sperm motility test results Table 2-6 Statistics of sperm motility test results

[0062] After the same semen was processed, the semen motility of the experimental group exceeded that of the control group, and the difference was significant.

[0063] 2.3.7. Statistics of sperm storage time experimental results Table 2-7 Statistics of sperm storage time experimental results

[0064] According to the general requirements of the pig farming industry, sperm motility should not be less than 70% before insemination. The sperm preservation reagent in the experimental group was significantly extended, exceeding the control group by 48 hours, with a significant difference.

[0065] 2.3.8. Experiment on treatment of unqualified semen Table 2-8 Statistics of unqualified semen treatment results

[0066] After the treatment of the unqualified semen, the sperm motility was significantly improved. Fig.11 , Fig.12 .

[0067] 2. Experimental Results The experimental results show that the device and method described in this application have a significant effect on improving sperm motility and sperm storage time, and can also meet qualified standards after processing unqualified semen.

[0068] 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 it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, 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 device for improving the storage time and sperm motility of pig semen, characterized in that: The invention comprises a filter cup (1), a primary microporous filter membrane (2), a drainage bag (3), a sedimentation cup (4), a secondary microporous filter membrane module (5), a collection cup (6) and an adjustable negative pressure electric suction device (7), wherein the bottom of the filter cup (1) is connected to the sedimentation cup (4), the secondary microporous filter membrane module (5) is arranged at the bottom of the sedimentation cup (4), the drainage bag (3) and the cup mouth of the sedimentation cup (4) are sleeved, the primary microporous filter membrane (2) is arranged above the drainage bag (3), and the sedimentation cup (4) is further provided with an air suction port (42) and a flow guide port (43), wherein the air suction port (42) is connected to the collection cup (6) through a connecting pipe, and the flow guide port (43) is connected to the adjustable negative pressure electric suction device (7) through a connecting pipe.

2. The device for improving the storage time and sperm motility of pig semen according to claim 1, characterized in that: The filter cup (1) is a borosilicate glass barrel without a bottom. The diameter of the filter cup (1) is 10-15 cm. A fixing device is installed at one end of the filter cup (1), and an internal thread (11) is provided in the fixing device. The sedimentation cup (4) is provided with an external thread (41) used in conjunction with the internal thread (11).

3. The device for improving the storage time and sperm motility of pig semen according to claim 2, characterized in that: The thickness of the primary microporous filtration membrane (2) is 10-40 µm, and the diameter is the same as that of the filter cup (1). The micropore diameter of the primary microporous filtration membrane (2) is 40-60 µm, and the micropore spacing is 20-40 μm.

4. The device for improving the storage time and sperm motility of pig semen according to claim 1, characterized in that: The drainage bag (3) is made of polyethylene and has a thickness of 10-20 μm. The diameter of the drainage bag (3) is the same as that of the sedimentation cup (4), and the micropore spacing is 20-40 μm.

5. The device for improving the storage time and sperm motility of pig semen according to claim 1, characterized in that: The sedimentation cup (4) is a round cup made of borosilicate glass, and the diameter of the sedimentation cup (4) is consistent with the diameter of the filter cup (1); the air suction port (42) is located on the side of the sedimentation cup (4), 20-50 mm away from the cup mouth, and is arranged to protrude; the flow guide port (43) is located directly below the air suction port (42), 10-20 mm away from the cup bottom, and is arranged to protrude.

6. The device for improving the storage time and sperm motility of pig semen according to claim 1, characterized in that: The secondary microporous filtration membrane module (5) comprises a base (51) and a secondary microporous filtration membrane (52); the base (51) is made of polycarbonate, and the diameter of the base (51) is slightly smaller than the sedimentation cup (4); the secondary microporous filtration membrane (52) is a hydrophilic nylon mesh membrane.

7. The device for improving the storage time and sperm motility of pig semen according to claim 6, characterized in that: The secondary microporous filtration membrane (52) has a thickness of 10-40 µm and a micropore diameter of 20-30 µm. The diameter of the secondary microporous filtration membrane (52) is the same as that of the sedimentation cup (4), and the micropore spacing is 10-20 μm.

8. The device for improving the storage time and sperm motility of pig semen according to claim 1, characterized in that: The collecting cup (6) is a glass beaker, and the connecting tube is a plastic tube.

9. A method for improving the storage time and sperm motility of pig semen, characterized in that: Using the device for improving the storage time and sperm motility of pig semen as claimed in any one of claims 1 to 8, the method comprises the following steps: Step 1: Pre-semen collection physiological regulation, including the following sub-steps: Optimize the boars’ antioxidant capacity by feeding them a special feed supplemented with 0.1% L-arginine and 0.05% selenium yeast 48 hours before semen collection; 2 hours before semen collection, the boars were placed in a bionic lighting environment to simulate the lighting conditions before natural mating and stimulate testosterone secretion; After the physiological regulation of pre-semen collection is completed, the boar semen is collected; Step 2: After the boar semen is collected, the device is placed in a bionic environment simulation water bath pot, the water bath temperature is 37±0.5°C, and the water bath environment parameters are dynamically adjusted through the temperature control module, including: the carbon dioxide concentration simulating the environment in 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, an amplitude of 0.5-1 mm, and the adjustable negative pressure electric suction device (7) is started at the same time, the negative pressure value is set to -50 to -80 kPa, and is dynamically adjusted according to the semen flow rate; Step 3: Pour the fresh boar semen collected in step 1 into the filter cup (1). Under the action of negative pressure and gravity, the semen passes through the primary microporous filter membrane (2) to filter out colloid and impurities; Step 4: The semen that has passed through the primary microporous filtration membrane (2) flows along the drainage bag (3) onto the secondary microporous filtration membrane (52), and the dead sperm and fragmented impurities in the semen settle to the bottom. Sperm with deformed tails 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 settle for 30 minutes. Step 5: After the standing time is over, sperm is collected in stages through the diversion port (43), specifically: S501, in the first 10 min, the sperm with the strongest head activity that penetrates the secondary microporous filtration membrane (52) is collected by negative pressure suction; S502, for the next 20 minutes, pulsed negative pressure is used, with an interval of 5 seconds to open / close, to collect the remaining high-motility sperm, and the selected sperm are introduced into a collection cup (6) pre-filled with arginine-trehalose composite protective solution with concentrations of 0.5 mM and 5 mM, respectively, through a connecting tube, to form a directional laminar flow 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, wherein the diluent comprises the following components: L101, basic diluent: glucose 2.5g / L, sodium citrate 1.2g / L, EDTA 0.1g / L; L102, functional additives: melatonin 0.1µM, superoxide dismutase (SOD) 50U / mL, nano-sized α-tocopherol liposome 0.05%; Among them, the particle size of nano-sized α-tocopherol liposomes is 50-100nm; After mixing, pack into antifreeze polyethylene bags and store at programmed temperature: N101, first stage: from 37°C to 4°C at -0.5°C / min; N102, second stage: reduce the temperature from 4℃ to -196℃ at -5℃ / min, and store in liquid nitrogen for a long time.

Images