Additive of boar semen diluent and application method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of N-Acetyl-D-Glucosamine as an additive in a boar semen diluent preserved at 17 DEG C. The invention further relates to a preparation method of the N-Acetyl-D-Glucosamine. By adding 2.5 mM of N-Acetyl-D-Glucosamine into the boar semen diluent, the activity and the forward movement proportion of boar sperms can be remarkably improved under the preservation condition of 17 DEG C, and meanwhile, no adverse effect is caused on the lipid oxidation state and acrosome integrity of the boar sperms. The method is easy and convenient to operate, low in cost, suitable for the artificial insemination process of a large-scale pig farm and capable of effectively prolonging the preservation time of the boar semen and improving the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an additive for diluent of pig semen stored at 17℃ and an application method thereof. BACKGROUND

[0002] Pig artificial insemination technology has been widely used in large-scale pig farms, effectively improving production efficiency and economic benefits. However, this technology has high requirements for boar semen quality, and semen storage quality directly affects the pregnancy rate of sows and the number of piglets. At present, commercial boar stations usually mix raw semen with diluent in proportion to produce semen products of different specifications, which are transported and sold at 17℃. Semen diluent can provide energy and suitable environment for sperm survival, so improving the diluent formula is of great significance for extending the storage time of semen, maintaining sperm motility and promoting the popularization of artificial insemination technology.

[0003] N-Acetyl-D-Glucosamine is a naturally occurring amino sugar that can participate in the regulation of cellular stress response. When cells encounter oxidative stress, heat shock, nutrient deficiency, DNA damage and other pressures, the modification level of glycosylated N-Acetyl-D-Glucosamine often increases rapidly, helping cells adapt to the stress environment by regulating the function of key proteins. Previous studies have shown that the content of N-Acetyl-D-Glucosamine in the semen of patients with primary infertility is lower than that of normal people, indicating that it has a potential impact on semen quality. The effect of adding N-Acetyl-D-Glucosamine to pig diluent on the storage of pig semen at 17℃ is not recorded in the prior art. SUMMARY

[0004] The purpose of the present application is to provide an additive for diluent of pig semen stored at 17℃, which can maintain and improve sperm motility during the storage of pig semen.

[0005] Another purpose of the present application is to determine the application method of the additive.

[0006] The technical solution of the present application is as follows:

[0007] An additive for diluent of pig semen stored at 17℃ is N-Acetyl-D-Glucosamine.

[0008] Preferably, the additive is added to the diluent of pig semen, and the concentration of the diluent in the pig semen after dilution is 55.3 mg / mL, i.e. the concentration is 2.5 mM.

[0009] The application method of the aforementioned additive for diluent of pig semen stored at 17℃ comprises the following steps:

[0010] S1: 0.553 g N-Acetyl-D-Glucosamine powder was dissolved in 10 mL commercial semen diluent to prepare N-Acetyl-D-Glucosamine concentrated stock solution, which was stored at -20℃ for later use.

[0011] S2: The commercial pig semen diluent was preheated to 37℃, and then fresh pig semen was mixed with the commercial pig semen diluent according to the production requirements to prepare diluted pig semen. After adding the N-Acetyl-D-Glucosamine concentrated stock solution and mixing, the diluted pig semen was stored at 17℃.

[0012] Preferably, in step S2 of the application method, 10 mL of the N-Acetyl-D-Glucosamine concentrated stock solution prepared in step 1 is added to every 1 L of diluted pig semen.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The addition of N-Acetyl-D-Glucosamine during the dilution of pig semen can significantly improve the sperm motility of pig semen stored at 17℃ for 48 hours.

[0015] The addition of N-Acetyl-D-Glucosamine during the dilution of pig semen can significantly and significantly improve the proportion of forward movement of pig sperm within 48 and 96 hours of storage at 17℃.

[0016] The addition of N-Acetyl-D-Glucosamine during the dilution of pig semen has no negative effect on the lipid oxidation level and acrosome integrity of pig sperm, is safe, and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Statistical chart of the effect of N-Acetyl-D-Glucosamine on pig sperm motility in the examples.

[0018] Figure 2 Statistical chart of the effect of N-Acetyl-D-Glucosamine on the proportion of forward movement of pig sperm in the examples.

[0019] Figure 3 Statistical chart of the effect of N-Acetyl-D-Glucosamine on the lipid oxidation level of pig sperm in the examples.

[0020] Figure 4 Statistical chart of the effect of N-Acetyl-D-Glucosamine on the acrosome integrity of pig sperm in the examples. DETAILED DESCRIPTION

[0021] The application will be further described below in connection with the drawings and specific examples, which are only used to illustrate the application and not to limit the protection scope of the application. Those skilled in the art can make equivalent modifications or adjustments according to the content of the application, and these modifications are also considered to fall within the protection scope of the application.

[0022] Unless otherwise specified, the experimental methods used in the examples are conventional methods in the art, and the materials and reagents used can be obtained through commercial channels.

[0023] The pig semen used in the examples is derived from commercial boar stations.

[0024] To verify the effect of N-Acetyl-D-Glucosamine on the quality of pig semen, the following experiments were carried out:

[0025] I. Screening of suitable concentration of N-Acetyl-D-Glucosamine for improving semen quality

[0026] Commercial pig semen diluent (Androstar Plus, Minitube) was used to prepare the working solution. 10 mL of diluent was used to dissolve 0.553 g of N-Acetyl-D-Glucosamine powder to prepare a concentrated stock solution of N-Acetyl-D-Glucosamine. Freshly collected pig semen was diluted with diluent and divided into 4 parts, each containing 1 mL. 0, 10, 20, and 30 μL of N-Acetyl-D-Glucosamine concentrated stock solution were added respectively, and mixed well to make the final concentration of 0 (control group), 2.5, 5, and 7.5 mM, respectively. At 0 h, 48 h, and 96 h after semen collection, 100 μL of sample was preheated at 37°C for 1 minute, and the remaining sample was stored at 17°C. 3 μL of preheated semen sample was taken to a four-chamber slide, and Prism Hexian Digital Agriculture semen analysis system was used to detect sperm motility and forward movement ratio. The average value of three fields was observed for each sample.

[0027] A total of 23 boar semen samples were measured for sperm motility and forward movement ratio at different N-Acetyl-D-Glucosamine concentrations at 0 h, 48 h, and 96 h. As shown in Figure 1 the results showed that compared with the control group, the addition of 2.5 mM N-Acetyl-D-Glucosamine can significantly improve the sperm motility at 48 h. As shown in Figure 2As shown, the addition of 2.5 mM N-Acetyl-D-Glucosamine significantly increased the proportion of sperm with progressive motility at 48 h and significantly increased it at 96 h. Therefore, 2.5 mM is the optimal concentration of N-Acetyl-D-Glucosamine for improving semen preservation quality.

[0028] II. Effects of N-Acetyl-D-Glucosamine on Sperm Lipid Oxidation

[0029] Diluted semen was treated with 2.5 mM N-Acetyl-D-Glucosamine, and the degree of sperm lipid oxidation was detected at 96 h. Semen samples were centrifuged at 600×g for 5 min, the supernatant was discarded, and the precipitate was resuspended in PBS buffer and washed twice, finally resuspended in 100 μL PBS. The Beyotime lipid peroxidation assay kit was used, and BODIPY 581 / 591 C11 staining solution was added to the sperm suspension to achieve a cell density of [missing information]. Incubate at 37°C in the dark for 20 min. After incubation, centrifuge and wash twice, resuspend, and observe under a fluorescence microscope.

[0030] C11 (581 / 591 nm) emits red fluorescence in the reduced state (Ex / Em = 581 / 591 nm) and turns green fluorescence after oxidation (Ex / Em ≈ 488 / 510 nm). The percentage of sperm emitting red fluorescence was used to evaluate lipid oxidation levels. Boar sperm plasma membranes are rich in polyunsaturated fatty acids, making them susceptible to oxidative damage. This damage is detrimental to maintaining the integrity of sperm structure and function, thus weakening sperm motility and even causing infertility. Results showed no significant difference in sperm lipid oxidation levels between the 2.5 mM N-Acetyl-D-Glucosamine treatment group and the control group. (See [link to relevant documentation]). Figure 3 This indicates that N-Acetyl-D-Glucosamine does not affect the oxidative state of sperm lipids.

[0031] III. Effects of N-Acetyl-D-Glucosamine on Sperm Acrosome Integrity

[0032] Diluted semen was treated with 2.5 mM N-Acetyl-D-Glucosamine, and acrosome integrity was assessed at 96 h. Semen samples were centrifuged, washed, and resuspended in 100 μL PBS. DAPI (1 μg / mL) was added and incubated in the dark for 5 min. After washing, sperm smears were prepared, fixed with 4% paraformaldehyde for 5 min, and washed with PBS. Staining was performed using the GENMED sperm acrosome morphology peanut lectin fluorescent labeling (PNA-FITC) kit, incubated at room temperature in the dark for 20 min, washed, and then mounted with an anti-quenching agent for observation under a fluorescence microscope.

[0033] FITC-PNA is typically excited in the 450-500 nm range, emitting green fluorescence. Intact acrosomes exhibit a distinct green fluorescence signal, indicating that sugar molecules on the acrosome membrane bind to FITC-PNA; damaged acrosome structures or incomplete sugar molecule exposure result in no green fluorescence or a weak fluorescence signal. The proportion of intact acrosomes is obtained by calculating the percentage of sperm exhibiting green fluorescence out of the total number of DAPI-stained sperm. Acrosome integrity is a prerequisite for sperm to undergo the acrosome reaction, which is the only way for sperm to penetrate the zona pellucida of the egg, directly determining whether sperm possess fertilization capacity. Therefore, acrosome integrity is a key factor in predicting sperm fertilization capacity and ensuring reproductive efficiency. Results showed no significant difference in acrosome integrity between the 2.5 mM N-Acetyl-D-Glucosamine treatment group and the control group. (See [link to relevant documentation]). Figure 4 This indicates that N-Acetyl-D-Glucosamine has no adverse effect on sperm acrosome structure.

[0034] In conclusion, adding 2.5 mM N-Acetyl-D-Glucosamine to porcine semen diluent significantly improves sperm motility at 17°C for 48 hours, significantly improves the proportion of sperm with progressive motility at 48 hours, and significantly improves the proportion of sperm with progressive motility at 96 hours, without affecting sperm lipid oxidation stability or acrosome integrity. This additive is simple to prepare and use, suitable for existing production processes, and has positive implications for improving the efficiency of artificial insemination in pigs and increasing enterprise profits.

[0035] The above embodiments are merely illustrative of the technical solutions of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art without departing from the principles of the present invention should be covered within the protection scope of the present invention.

Claims

1. An additive for a diluent used to preserve bovine semen at 17°C, characterized in that, The additive is N-Acetyl-D-Glucosamine.

2. The additive according to claim 1, characterized in that: The additive was added to a commercially available porcine semen diluent, and its concentration in the diluted porcine semen was 55.3 mg / mL, or 2.5 mM.

3. The method of applying the additive in the diluent of any of the aforementioned boar semen, characterized in that, Includes the following steps: S1 uses 10 mL of commercial boar semen diluent to dissolve 0.553 g of N-Acetyl-D-Glucosamine powder to prepare N-Acetyl-D-Glucosamine concentrated stock solution, which is stored at -20℃ for later use; S2 preheats the commercial boar semen diluent to 37°C, then mixes the fresh boar semen and the commercial boar semen diluent in a specific ratio according to production requirements to prepare diluted boar semen. After mixing the diluted semen with the aforementioned N-Acetyl-D-Glucosamine concentrate, it is stored at 17°C.

4. The application method according to claim 3, characterized in that: In step 2, add 10 mL of the N-Acetyl-D-Glucosamine concentrate from step 1 to every 1 L of diluted bovine semen.

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