Application of BSA (Bovine Serum Albumin) in improving performance of 500 million 60 ml low-density boar semen

Abstract

The invention discloses application of BSA (Bovine Serum Albumin) in improving the performance of low-density boar semen, and the BSA is particularly suitable for 0.5 billion / 60ml low-density boar semen. Accordingly, the inventor also develops a boar semen diluent, which comprises a basic solution, a nutritional agent, a buffer agent and an electrolyte, and also comprises a core film protecting agent, an antioxidant and a composite antibacterial agent, the core film protecting agent is BSA, the nutritional agent is composed of glucose and fructose, the antioxidant is composed of glutathione and arbutin, and the composite antibacterial agent is composed of penicillin, streptomycin and gentamicin. By accurately optimizing the BSA addition concentration and cooperating with glucose and fructose (dual-energy supply), a high-quality sperm protection microenvironment system of'targeted film protection-environment stabilization-energy supply 'is constructed, the problems of short preservation time and poor sperm form integrity when low-density sperms are treated by conventional diluents are solved, and cost reduction and efficiency improvement of large-scale pig farms can be realized.

Description

Technical Field

[0001] This invention belongs to the field of porcine artificial insemination technology, and particularly relates to the application of BSA in improving the performance of 500 million 60 ml of low-density porcine semen. Background Technology

[0002] In the practice of artificial insemination in large-scale pig farming, the industry has long used a conventional semen density of 1.2 billion / 60ml. While this method ensures reproductive efficiency, it suffers from low utilization of the original semen, indirectly leading to higher farming costs. To overcome this limitation, the industry has attempted to reduce the semen density to 500 million / 60ml (achieving higher utilization efficiency through dilution). However, conventional diluents have two major drawbacks that directly restrict the widespread application of low-density semen:

[0003] 1. Short storage time: In low-density environments, the "mutual protection" between sperm is significantly weakened. Commercially available diluents cannot resist the oxidative stress and osmotic pressure fluctuations of low-density boar semen. Within 120 hours of refrigeration at 17 ℃, sperm motility drops to about 60%, and after 144 hours, motility is ≤ 50%, which is far from meeting the requirements for semen storage time in cross-regional transportation and batch insemination.

[0004] 2. Poor integrity of sperm internal structure: Conventional diluents lack targeted membrane protection components. In low-density semen, sperm cell membranes are easily damaged by environmental stimuli, and acrosomes are easily detached. Within 120 hours of storage, the acrosome damage rate is ≥40% and the plasma membrane damage rate is ≥20%, which directly affects the conception rate of sows and creates a phenomenon of "reduced density and reduced efficiency".

[0005] In existing technologies, while BSA is used for membrane protection of biological agents, it suffers from significant compatibility defects due to the lack of specific optimization for low-density porcine semen systems. This makes it difficult to effectively repair the fragile sperm membrane structure in this type of semen, thus failing to simultaneously overcome the dual technical challenges of "insufficient preservation time" and "poor sperm internal structural integrity" in the preservation of low-density porcine semen. Therefore, developing a targeted improvement technology based on BSA has become crucial to breaking through the performance bottleneck of low-density semen and achieving "density reduction + efficiency maintenance + cost reduction". Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an application of BSA in improving the performance of 500 million 60ml low-density boar semen, thereby improving the preservation time and sperm morphology integrity of low-density boar semen, so as to achieve cost reduction and efficiency improvement in large-scale pig farms.

[0007] To solve the above technical problems, the present invention adopts the following technical solution:

[0008] Application of BSA (bovine serum albumin) in improving the performance of low-density bovine semen.

[0009] The density of low-density boar semen is 500 million / 60ml.

[0010] BSA is used as a core protective film agent in porcine semen diluent.

[0011] The boar semen diluent includes a base solution, nutrients, buffers and electrolytes, as well as a core protective agent, antioxidants and compound antibacterial agents; the core protective agent is BSA, the nutrients are composed of glucose and fructose, the antioxidants are composed of glutathione and arbutin, and the compound antibacterial agents are composed of penicillin, streptomycin and gentamicin.

[0012] The boar semen diluent uses a base solution as the solvent, which is ultrapure water; each 1L of boar semen diluent contains

[0013] Nutrients: 6-11g glucose, 2-5g fructose;

[0014] Core protective film agent: 0.3–5 g;

[0015] Buffers and electrolytes: potassium chloride 0.2-1g, sodium citrate 5-10g, sodium bicarbonate 0.3-2g, ethylenediaminetetraacetic acid 1-4g, tris(hydroxymethyl)aminomethane 3-8g;

[0016] Antioxidants: glutathione 1-5g, arbutin 10-18g;

[0017] Compound antibacterial agent: penicillin 0.2-0.3g, streptomycin 0.5-0.8g, gentamicin 0.1-0.8g.

[0018] The above-mentioned method for preparing boar semen diluent involves adding nutrients, buffers and electrolytes, antioxidants and compound antibacterial agents to a base solution and mixing them thoroughly. Then, a core protective film agent is added and the mixture is stirred and mixed until the desired solution is obtained.

[0019] The above preparation method involves accurately weighing 1000 mL of ultrapure water and placing it in a sterile beaker. Nutrients, buffers and electrolytes, antioxidants and compound antibacterial agents are added to the ultrapure water. The mixture is stirred with a magnetic stirrer at a speed of 50–130 r / min until completely dissolved. Then, the core protective film agent is added and the mixture is stirred until homogeneous to obtain the diluted boar semen solution.

[0020] The above method of using the boar semen diluent involves preheating the boar semen diluent, then adding the boar semen and diluting at a moderate temperature.

[0021] The above method involves heating the diluted boar semen solution in a water bath; selecting fresh boar semen, and then slowly adding the preheated diluted boar semen solution according to the measured amount, while maintaining the temperature of the mixing system to obtain diluted boar semen.

[0022] Heat in a water bath to 33 ℃; fresh boar semen has a sperm motility ≥80%, a semen density ≥100 million / mL, and a deformity rate <20%. Maintain the temperature of the mixing system at 32-35 ℃ and dilute the boar semen to a density of 500 million / 60mL.

[0023] Addressing the industry pain points of low-density semen, such as fragile sperm membranes, sensitivity to oxidative stress, short shelf life, poor sperm morphology integrity, and low utilization rate of conventional density semen, the inventors proposed using BSA (bovine serum albumin) to improve the performance of low-density boar semen, particularly suitable for 500 million / 60ml low-density boar semen. Based on this, the inventors also developed a boar semen diluent, including a base solution, nutrients, buffers, and electrolytes, as well as a core protective agent, antioxidants, and a compound antibacterial agent. The core protective agent is BSA, the nutrients consist of glucose and fructose, the antioxidants consist of glutathione and arbutin, and the compound antibacterial agent consists of penicillin, streptomycin, and gentamicin. By precisely optimizing the BSA concentration and synergistically combining glucose and fructose (dual energy supply), a high-quality sperm-protecting microenvironment system of "targeted protective membrane - environmental stability - energy supply" was constructed, solving the problems of short shelf life and poor sperm morphology integrity when treating low-density semen with conventional diluents. Compared with existing technologies, the beneficial effects of this invention are as follows:

[0024] 1. Addressing the pain point of short preservation time for 500 million / 60mL boar semen.

[0025] Using this invention, after diluting boar semen and storing it at 17 ℃ for 168 h, the sperm motility is still ≥70%, which fully meets the requirements for semen preservation time for cross-site transportation and batch insemination.

[0026] 2. Significantly improves sperm morphological integrity

[0027] After being stored at 17 ℃ for 168 h, the integrity of the acrosome and plasma membrane was ≥75%, and the deformity rate was <20%, effectively avoiding sperm inactivation caused by membrane damage and acrosome shedding, fundamentally solving the problem of "reduced density and reduced efficiency";

[0028] 3. Simultaneously achieve cost reduction and efficiency improvement

[0029] By improving the performance stability of low-density semen, the utilization rate of raw semen can be effectively increased, the demand for breeding boars can be reduced, and the costs of introducing breeding stock, feeding and disease prevention and control in large-scale pig farms can be reduced, which combines technological innovation and economic practicality.

[0030] 4. Easy to use and promote

[0031] The raw materials for the diluent (BSA, glucose, etc.) are all conventional reagents in the livestock industry and are easy to purchase; the preparation process does not require special equipment, is compatible with the existing artificial insemination process of large-scale pig farms, and can be quickly implemented. Attached Figure Description

[0032] Figure 1 The graph shows the effects of different preservatives on 500 million / 60ml boar semen. In the graph: A and B are the results of boar semen motility and deformity rate with added BSA, C and D are the results of boar semen motility and deformity rate with added plant polysaccharides, and E and F are the results of boar semen motility and deformity rate with added arginine.

[0033] Figure 2 The figure shows the sperm quality test results after adding different concentrations of BSA to the porcine semen diluent of this invention. In the figure: A is motility, B is high membrane potential sperm, C is plasma membrane integrity, and D is acrosome integrity. Detailed Implementation

[0034] Example 1: Effects of adding different preservatives on 500 million / 60ml boar semen

[0035] 1. Preparation of different diluents

[0036] 1.1 The porcine semen diluent of the present invention

[0037] Formulation: The porcine semen diluent of this invention comprises a base solution, nutrients, buffers, and electrolytes, as well as a core protective agent, an antioxidant, and a compound antibacterial agent. The core protective agent is BSA, the nutrients consist of glucose and fructose, the antioxidant consists of glutathione and arbutin, and the compound antibacterial agent consists of penicillin, streptomycin, and gentamicin. The concentration ratios and functions of each component in this example are shown in Table 1 below.

[0038]

[0039] Preparation: Accurately weigh 1000 mL of ultrapure water and place it in a sterile beaker. Add the nutrients, buffers and electrolytes, antioxidants, and compound antibacterial agents to the ultrapure water. Use a magnetic stirrer to stir at 50–130 r / min until completely dissolved (avoid generating bubbles). Then add the core protective film agent and continue stirring to mix thoroughly (ensuring complete dissolution of BSA without any flocculent matter). This yields the diluted boar semen solution. Before use, heat to 33°C in a water bath and use within 2 hours or store at 17°C for later use. Prepare as needed.

[0040] The YX diluent used below is the boar semen diluent of the present invention without the addition of BSA, and the preparation method is basically the same as above.

[0041] 1.2 Preparation of diluent from commercial diluent powder

[0042] Strictly follow the instructions to ensure accurate concentration and complete dissolution, so as to achieve the best performance of the diluent.

[0043] 2. Methods for adding different protective agents:

[0044] After adding equal amounts of BSA, plant polysaccharide, and arginine to the various dilutions in step 1, mix them evenly and let them stand for 10-15 minutes, then filter them with a 0.22μm bacterial filter to remove bacteria.

[0045] BSA has a molecular weight of 66.4 kDa and a purity of ≥98%. It is filtered through a 0.22μm sterile filter membrane before use to prevent microbial contamination that could damage sperm morphology.

[0046] Plant polysaccharides, specifically licorice polysaccharides with a purity >95%.

[0047] 3. Semen dilution method:

[0048] Fresh, high-quality boar semen (sperm motility ≥80%, semen density ≥100 million / mL, abnormality rate <20%) was selected. Different diluted solutions with added preservatives were slowly injected into fresh boar semen samples to obtain 500 million / 60ml diluted semen. The diluted semen was then allowed to cool naturally at room temperature until it reached approximately 19℃, at which point it was stored in a 17℃ constant-temperature refrigerator. Semen samples were collected on days 1, 3, 5, and 7 after dilution for semen quality testing.

[0049] 4. Semen quality testing

[0050] The diluted semen was removed from a 17°C constant temperature freezer, repeatedly inverted and shaken to mix. 2 μl of semen was placed on a preheated 39°C glass slide and heated for another 3 minutes. Sperm motility, progressive motility, and abnormality rate were measured using a CASA system. At least 500 sperm were analyzed in each sample group. Results are as follows: Figure 1 As shown.

[0051] Figure 1 middle,

[0052] A: The horizontal axis represents time, and the vertical axis represents sperm motility. Black bars represent YX diluent + BSA, serving as the control group. Yellow bars represent other brands of commercial diluent 1 + BSA. Blue bars represent other brands of commercial diluent 2 + BSA.

[0053] B: The horizontal axis represents time, and the vertical axis represents the distortion rate. Black bars represent YX diluent + BSA, serving as the control group. Yellow bars represent other brands of commercial diluent 1 + BSA. Blue bars represent other brands of commercial diluent 2 + BSA.

[0054] C: The horizontal axis represents time, and the vertical axis represents sperm motility. Black bars represent YX diluent + plant polysaccharides, serving as the control group. Yellow bars represent other brands of commercial diluent 1 + plant polysaccharides. Blue bars represent other brands of commercial diluent 2 + plant polysaccharides.

[0055] D: The horizontal axis represents time, and the vertical axis represents the distortion rate. Black bars represent YX dilution + plant polysaccharides, serving as the control group. Yellow bars represent other brands of commercial dilution powder 1 + plant polysaccharides. Blue bars represent other brands of commercial dilution powder 2 + plant polysaccharides.

[0056] E: The horizontal axis represents time, and the vertical axis represents sperm motility. Black bars represent YX diluent + arginine, serving as the control group. Yellow bars represent other brands of commercial diluent 1 + arginine. Blue bars represent other brands of commercial diluent 2 + arginine.

[0057] F: The horizontal axis represents time, and the vertical axis represents the deformity rate. Black bars represent YX dilution + arginine, serving as the control group. Yellow bars represent other brands of commercial dilution powder 1 + arginine; blue bars represent other brands of commercial dilution powder 2 + arginine. "ns" indicates no significant difference, "*" indicates a significant difference, "**" indicates an extremely significant difference, and "***" indicates an extremely significant difference. The number of boars was n = 15.

[0058] Figure 1 The results showed that the overall semen motility level was higher in all groups that added BSA. Figure 1 A): Among them, the 500 million / 60ml boar semen treated with YX diluent + BSA (i.e., this invention) showed significantly higher sperm motility than the other two commercially available diluent groups from the third day of refrigeration at 17°C; by the seventh day of storage, its sperm motility was still ≥60%, fully meeting the national standard requirements for sperm motility in artificial insemination. In contrast, the sperm motility of the groups with added plant polysaccharides or arginine was <60% by the seventh day. Figure 1 C and E) did not meet the national standards applicable to artificial insemination. This further confirms the unique advantages of BSA as a core protective agent in improving the long-term cold storage performance of 500 million / 60ml boar semen, with significantly better effects than common protective agents such as plant polysaccharides and arginine, as well as protective components in conventional commercial diluents. Furthermore, after treatment with different diluents and protective agents, there was no significant difference in the sperm abnormality rate of 500 million / 60ml boar semen. Figure 1 (B, D, F) indicates that after reducing semen density, none of the above treatment methods will have an adverse effect on the external morphology of sperm.

[0059] Example 2: Diluting and preserving boar semen with different concentrations of BSA added to YX diluent.

[0060] Referring to Example 1, different concentrations of BSA (0, 0.005%, 0.01%, 0.02%, 0.03%) were added to the boar semen diluent to dilute the boar semen, resulting in a diluted boar semen concentration of 500 million BSA per 60 ml. The diluted semen was then allowed to cool naturally at room temperature until it reached approximately 19°C, at which point it was stored in a 17°C constant-temperature refrigerator. The semen was collected on days 1, 3, 5, and 7 after dilution to test its antioxidant capacity.

[0061] Sperm motility test: Semen was taken from a 17°C constant temperature freezer, gently inverted and shaken repeatedly, and 2 μl of semen was heated on a glass slide preheated at 39°C for 4 min. Sperm motility was tested under the CASA system. At least 500 sperm were tested in each sample group.

[0062] Detection of high membrane potential sperm percentage: The dye stock solution was prepared according to the ratio of JC-1(200x):JC-1(5x):PBS = 1:40:160; then the dye stock solution was diluted 8 times with PBS, vortexed and mixed, and 195 μL / well was added to a 96-well plate in the dark; 5 μL of the semen sample was added to each well after gentle shaking, and incubated at 37°C in the dark for 20 min. The red light (JC-1 multiple bodies, high membrane potential) and green light (JC-1 single bodies, low membrane potential) channels were detected by flow cytometry. 10,000-20,000 sperm were analyzed, and the percentage of high membrane potential sperm was expressed as a red staining percentage.

[0063] Plasma membrane integrity: Prepare SYBR14 working solution first at a ratio of SYBR14:DMSO = 1:50, then prepare dye at a ratio of SYBR14 working solution:PI:PBS = 1:1:198. Take 5 μL of the semen sample after gentle shaking and add it to 195 μL of dye. Incubate at 37°C in the dark for 10 min. Wash twice with PBS and centrifuge (800 ×g, 8 min). Resuspend the sperm in 200 μL of PBS. Measure the red (PI) and green (SYBR14) channels using flow cytometry. Detect the sperm count of 10,000-20,000 sperm and count the proportion of sperm with intact plasma membranes.

[0064] Acrosome integrity: The working solution was prepared according to the ratio of FITC:PI:PBS = 0.5:1:196.5 and added to a 96-well plate at 195 μL / well in the dark. After gentle mixing, 5 μL of the semen sample was added to each well and incubated at 37°C in the dark for 10-20 min. After washing twice with PBS, the sample was centrifuged (800 g, 8 min) and resuspended in 200 μL of PBS. The red (PI) and green (FITC) channels were measured using flow cytometry. 10,000-20,000 sperm cells were detected, and the proportion of sperm cells with intact acrosomes was counted.

[0065] Test results as follows Figure 2 As shown. Figure 2 middle,

[0066] A: The horizontal axis represents storage time, and the vertical axis represents sperm motility. Black bars represent YX diluent + 0% BSA, serving as the control group. Yellow bars represent YX diluent + 0.005% BSA. Blue bars represent YX diluent + 0.01% BSA. Pink bars represent YX diluent + 0.02% BSA. Gray bars represent YX diluent + 0.03% BSA.

[0067] B: The horizontal axis represents time, and the vertical axis represents the percentage of sperm with high membrane potential. Black bars represent YX dilution + 0% BSA, serving as the control group. Yellow bars represent YX dilution + 0.005% BSA. Blue bars represent YX dilution + 0.01% BSA. Pink bars represent YX dilution + 0.02% BSA. Gray bars represent YX dilution + 0.03% BSA.

[0068] C: The horizontal axis represents time, and the vertical axis represents the percentage of sperm with intact plasma membranes. Black bars represent YX dilution + 0% BSA, serving as the control group. Yellow bars represent YX dilution + 0.005% BSA. Blue bars represent YX dilution + 0.01% BSA. Pink bars represent YX dilution + 0.02% BSA. Gray bars represent YX dilution + 0.03% BSA.

[0069] D: The horizontal axis represents time, and the vertical axis represents the percentage of sperm with intact acrosomes. Black bars represent YX dilution + 0% BSA, serving as the control group. Yellow bars represent YX dilution + 0.005% BSA. Blue bars represent YX dilution + 0.01% BSA. Pink bars represent YX dilution + 0.02% BSA. Gray bars represent YX dilution + 0.03% BSA. "ns" indicates no significant difference, "*" indicates a significant difference, "**" indicates an extremely significant difference, and "***" indicates an extremely significant difference. The number of boars is n = 10.

[0070] Figure 2 shows that after adding different concentrations of BSA, the sperm motility of 500 million / 60ml boar semen samples stored at 17℃ for 7 days was still >60%, with the group adding 0.01% BSA showing the best results. Figure 2 A). In the groups supplemented with 0.01% BSA and 0.02% BSA, the proportion of high membrane potential sperm was significantly higher from day 5 of storage compared to the group without BSA supplementation. Figure 2 B). Furthermore, from day 3 of storage, the group with added 0.01% BSA showed significantly higher plasma membrane and acrosome integrity than the group without added BSA. Figure 2 C, D).

[0071] The above results confirm that adding 0.01% BSA to 500 million / 60ml boar semen can effectively maintain sperm motility while ensuring the integrity of the sperm's internal structure and functional activity, providing key support for the long-term cold storage and artificial insemination applicability of this specification of boar semen.

Claims

1. Application of BSA in improving the performance of low-density pig semen.

2. Use according to claim 1, characterized in that: The density of the low-density pig semen is 500 million / 60ml.

3. BSA is used as a core protective agent in pig semen diluent.

4. A porcine semen diluent comprising a base fluid, a nutrient, a buffer and an electrolyte, characterized in that The core protective agent is BSA, the nutrient agent consists of glucose and fructose, the antioxidant agent consists of glutathione and arbutin, and the composite antibacterial agent consists of penicillin, streptomycin and gentamicin.

5. The porcine semen diluent of claim 4, wherein: The pig semen diluent takes ultrapure water as a solvent, and contains Nutrient agent: 6-11g of glucose and 2-5g of fructose; Core protective agent: 0.3-5g; Buffering agent and electrolyte: 0.2-1g of potassium chloride, 5-10g of sodium citrate, 0.3-2g of sodium bicarbonate, 1-4g of ethylenediaminetetraacetic acid and 3-8g of tris-hydroxymethyl aminomethane; Antioxidant agent: 1-5g of glutathione and 10-18g of arbutin; Composite antibacterial agent: 0.2-0.3g of penicillin, 0.5-0.8g of streptomycin and 0.1-0.8g of gentamicin.

6. The method of claim 4, wherein the preparation of the pig semen diluent is characterized by: The nutrient agent, buffering agent and electrolyte, antioxidant agent and composite antibacterial agent are added into the base solution and mixed uniformly, and then the core protective agent is added and stirred uniformly to obtain the pig semen diluent.

7. The method of claim 6, wherein: 1000ml of ultrapure water is accurately weighed and placed in a sterile beaker, and the nutrient agent, buffering agent and electrolyte, antioxidant agent and composite antibacterial agent are added into the ultrapure water; a magnetic stirrer is used to stir at a speed of 50-130r / min until complete dissolution, and then the core protective agent is added and stirred uniformly to obtain the pig semen diluent.

8. The method of using the boar semen diluent of claim 4, wherein: The pig semen diluent is preheated, and then the pig semen is added for isothermal dilution.

9. The method of use of claim 8, wherein: The pig semen diluent is heated without water, and fresh pig semen is selected, then the preheated pig semen diluent is slowly added into the pig semen according to the measurement, and the temperature of the mixed system is maintained to obtain the diluted pig semen.

10. The method of use of claim 7, wherein: The heating is to 33℃, the fresh pig semen has a sperm motility of ≥80%, a semen density of ≥100 million / mL and a deformity rate of <20%, the temperature of the mixed system is maintained at 32-35℃, and the density of the diluted pig semen is 500 million / 60ml.

Images