Additive of diluent for preserving boar semen at 17 DEG C and application method thereof

Abstract

The invention relates to an additive of a diluent for preserving boar semen at 17 DEG C and an application method thereof. Myoinositol with the concentration of 1 mg / mL is added into a boar semen diluent, so that the activity and forward movement proportion of boar sperms under the preservation condition of 17 DEG C can be remarkably improved, and meanwhile, the lipid oxidation state and acrosome integrity of the boar sperms are not influenced. The method is easy and convenient to operate, low in cost, suitable for the large-scale pig farm artificial insemination technology and beneficial to prolonging the pig semen preservation time and improving the breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an additive for a diluent for preserving bovine semen at 17°C and its application method. Background Technology

[0002] Artificial insemination in pigs has been widely applied in large-scale pig farms, effectively improving production efficiency and economic benefits. However, this technology requires high-quality boar semen, as the quality of semen preservation directly affects sow conception rate and litter size. Currently, commercial boar pens typically mix raw semen with diluent in a specific ratio to produce semen products of different specifications, which are then transported and sold at a constant temperature of 17°C. Semen diluent provides sperm with the energy and suitable environment necessary for survival; therefore, improving the diluent formulation is of great significance for extending semen preservation time, maintaining sperm motility, and promoting the widespread adoption of artificial insemination technology.

[0003] Myoinositol is a naturally occurring glycoside whose phosphorylated product is an important intracellular second messenger involved in the signal transduction of various hormones, including insulin and follicle-stimulating hormone. Existing research indicates that myoinositol can aid in the functional recovery of frozen semen after thawing; however, its application in the preservation of porcine semen at 17°C and in corresponding diluents has not been reported. Summary of the Invention

[0004] The purpose of this invention is to provide an additive for a diluent for preserving bovine semen at 17°C, which can maintain or improve sperm motility during the preservation of bovine semen.

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

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

[0007] Myoinositol is an additive for a diluent used to preserve bovine semen at 17°C.

[0008] Preferably, the additive is added to the boar semen diluent, and its concentration in the diluted boar semen is 1 mg / mL.

[0009] A method for applying an additive to a diluent for preserving bovine semen at 17°C includes the following steps:

[0010] S1 uses 10 mL of commercial boar semen diluent to dissolve 1 g of Myoinositol powder to prepare Myoinositol concentrated stock solution, which is stored at -20℃ for later use.

[0011] 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 with the aforementioned Myoinositol concentrated stock solution, it is stored at 17°C.

[0012] Preferably, in step S2 of the aforementioned application method, 10 mL of the Myoinositol concentrate described in step 1 is added to every 1 L of diluted bovine semen.

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

[0014] Adding Myoinositol during the dilution of boar semen can significantly improve sperm motility when stored at 17°C for 96 hours.

[0015] Adding Myoinositol during the dilution of boar semen can significantly increase the proportion of forward sperm motility in boar semen stored at 17°C for 48 and 96 hours.

[0016] Adding Myoinositol during the dilution of boar semen has no negative impact on the lipid oxidation level and acrosome integrity of boar sperm, has good safety, and has good prospects for widespread application. Attached Figure Description

[0017] Figure 1 This is a statistical chart illustrating the effect of Myoinositol on porcine sperm motility in the examples.

[0018] Figure 2 This is a statistical graph showing the effect of Myoinositol on the proportion of forward motility in porcine sperm in the examples.

[0019] Figure 3 This is a statistical graph showing the effect of Myoinositol on the lipid oxidation level of porcine sperm in the examples.

[0020] Figure 4 This is a statistical chart illustrating the effect of Myoinositol on the acrosome integrity of porcine sperm in the examples. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make equivalent improvements or adjustments based on the content of the present invention, and these modifications should also be considered to fall within the scope of protection of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the examples are conventional methods in the art, and the materials and reagents used are commercially available.

[0023] The boar semen used in the examples was obtained from commercial boar stations.

[0024] To verify the effect of Myoinositol on boar semen quality, the following experiment was conducted:

[0025] I. Screening of suitable concentrations of Myoinositol for improving semen quality

[0026] Working solutions were prepared using commercially available porcine semen diluent (Androstar Plus, Minitube). 10 mL of diluent was used to dissolve 1 g of Myoinositol powder, creating a concentrated Myoinositol stock solution. Freshly collected porcine raw semen was diluted with the diluent and dispensed into four 1 mL aliquots. 0, 10, 20, and 30 μL of concentrated Myoinositol stock solution were added to each aliquot, and the mixture was stirred to achieve final concentrations of 0 (control group), 1, 2, and 3 mg / mL, respectively. On the day of semen collection (0 h), 48 h, and 96 h, 100 μL of sample was preheated in a 37°C water bath for 1 minute, while the remaining samples were stored at 17°C. 3 μL of the preheated semen sample was transferred to a four-chamber slide, and sperm motility and the proportion of progressive motility were measured using the Prism Hexian Digital Agriculture semen analysis system. Three fields of view were observed for each sample, and the average value was taken.

[0027] Sperm motility and the proportion of progressively motile sperm were measured in semen samples from 23 boars at different Myoinositol concentrations at 0 h, 48 h, and 96 h. Figure 1 As shown, the results indicated that, compared with the control group, the addition of 1 mg / mL Myoinositol significantly improved sperm motility at 96 h. Figure 2 As shown, adding 1 mg / mL of Myoinositol significantly increased the proportion of sperm with progressive motility at 48 h and 96 h. Therefore, 1 mg / mL is the optimal concentration of Myoinositol for improving semen preservation quality.

[0028] II. Effects of Myoinositol on Sperm Lipid Oxidation

[0029] Diluted semen was treated with 1 mg / mL Myoinositol, 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 1 mg / mL Myoinositol treatment group and the control group. (See [link to relevant documentation]). Figure 3 This indicates that Myoinositol does not affect the lipid oxidation state of sperm.

[0031] III. Effects of Myoinositol on Sperm Acrosome Integrity

[0032] Diluted semen was treated with 1 mg / mL Myoinositol, 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 determined 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 crucial factor in predicting sperm fertilization capacity and ensuring reproductive efficiency. Results showed no significant difference in acrosome integrity between the 1 mg / mL Myoinositol treatment group and the control group. (See [link to relevant documentation]). Figure 4 This indicates that Myoinositol has no adverse effect on sperm acrosome structure.

[0034] In conclusion, adding 1 mg / mL Myoinositol to porcine semen diluent significantly improves the sperm motility ratio after storage at 17°C for 96 hours, and substantially increases the proportion of progressively motile sperm at 48 and 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 Myoinositol.

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

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 1 g of Myoinositol powder to prepare Myoinositol 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 with the aforementioned Myoinositol concentrated stock solution, 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 Myoinositol concentrate from step 1 to every 1 L of diluted bovine semen.

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