Additive to improve the reproductive performance of female animals

Abstract

This invention relates to the field of veterinary medicine, and more particularly to additives for enhancing the fertility of female animals. Research in this invention shows that decanal can increase the proliferative activity of ovarian granulosa cells, thus making it suitable as an additive to enhance the fertility of female animals. As an additive, decanal can increase the number of granulosa cells, especially S-phase cells, and has a significant impact on proliferation-related genes. Furthermore, decanal has previously been commonly used as a food additive and has good safety profiles.

Description

Technical Field

[0001] This invention relates to the field of animal medicine technology, and in particular to additives that enhance the fertility of female animals. Background Technology

[0002] Granulosa cells play a crucial role in the growth and development of follicles and the maturation of oocytes, providing a suitable microenvironment for both processes. Granulosa cells provide nutrients to oocytes and promote oocyte maturation. Therefore, enhancing the proliferative activity of bovine ovarian granulosa cells is an effective way to improve the fertility of cows.

[0003] Decanal, also known as decacarbonal or n-decanal, has the following structural formula:

[0004]

[0005] Decanal is a transparent to pale yellow liquid with a strong citrus peel odor. It can be used to formulate natural food flavorings such as orange, mandarin orange, and lemon. Currently, it is commonly used as a food flavoring in beverages, biscuits, pastries, frozen foods, candies, seasonings, dairy products, canned goods, and alcoholic beverages. The technology for synthesizing flavorings with decanal is highly mature, has a wide range of applications, and good safety performance.

[0006] While decanal is edible, its effects at the cellular level have not been fully studied, particularly its effects on ovarian granulosa cells. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an additive that enhances the fertility of female animals.

[0008] This invention provides the application of decanal in the preparation of formulations that enhance the fertility of female animals.

[0009] The additives that enhance the fertility of female animals include those that promote the proliferation of ovarian granulosa cells.

[0010] The promotion of ovarian granulosa cell proliferation includes:

[0011] Promotes the proliferation and activity of granulocytes;

[0012] Increase the number of S-phase cells in granulocytes;

[0013] And / or increase the expression levels of CDK1, CDC42, PCNA, cyclin A2 and / or cyclin D2 genes.

[0014] In this invention, the concentration of decanal, which promotes granulocyte proliferation, increases the number of granulocytes in S phase, or enhances the expression level of granulocyte proliferation-related genes, is 40-80 ng / ml, for example, 40 ng / ml, 50 ng / ml, 60 ng / ml, and / or 80 ng / ml. Preferably, it is 50 ng / ml.

[0015] The present invention also provides an additive to improve the fertility of female animals, characterized in that it comprises decanal and excipients.

[0016] The additives described in this invention are powders, granules, solutions, or suspensions.

[0017] The content of decanal in the additive of the present invention is 40~80 ng / ml. Preferably, the content of decanal is 50 ng / ml.

[0018] Furthermore, the present invention also provides a feed for improving the fertility of female animals, comprising the additives described above.

[0019] The feed described in this invention also includes a basic feed. The selection of the basic feed depends on the species of animal, and this invention does not limit this selection.

[0020] The present invention also provides a feeding method for improving the fertility of female animals, which includes administering the additives or feeds as described above.

[0021] In this invention, the female animal is a female even-toed ungulate, including but not limited to: Suidae, Hippopotamia, Maverickae, Camelidae, Mosquitoidae, Cervidae, Giraffeae, Pronghornidae, and / or Bovidae. In embodiments of this invention, Bovidae animals are used as test subjects for experiments, such as animals from the genera *Buffalo*, *Bos*, *African Buffalo*, or *Buffalo bison*. Preferably, the test subject is a cow, more specifically, the yellow cattle species.

[0022] This invention demonstrates that decanal can enhance the proliferative activity of ovarian granulosa cells, thus making it a suitable additive for improving the reproductive capacity of female animals. As an additive, decanal can increase the number of granulosa cells, particularly those in the S phase, and has a significant impact on proliferation-related genes. Furthermore, decanal has previously been commonly used as a food additive and has shown good safety profiles. Attached Figure Description

[0023] Figure 1 Example 1: Effect of decanal on the proliferation activity of bovine ovarian granulosa cells;

[0024] Figure 2 Example 2: Effect of decanal on the proliferation of bovine ovarian granulosa cells;

[0025] Figure 3Example 2: Effect of decanal on the S phase of bovine ovarian granulosa cells;

[0026] Figure 4 Example 3 illustrates the effect of decanal on genes related to the proliferation of bovine ovarian granulosa cells. Detailed Implementation

[0027] This invention provides an additive to enhance the fertility of female animals. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0028] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0029] The present invention will be further illustrated below with reference to the embodiments:

[0030] Example 1: Determination of the effect of decanal on the proliferation activity of bovine ovarian granulosa cells using the CCK-8 assay

[0031] Experimental materials: Bovine ovaries were collected from the slaughterhouse, and decanal (analytical standard, GC=98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0032] Experimental Methods: Primary bovine ovarian granulosa cells were extracted and seeded at 5000 cells / well in 96-well plates. After cell adhesion, 200 μL of decanal (DMEM with fetal bovine serum) at different drug concentrations was added to each well. After 48 hours of drug treatment, 200 μL of CCK-8 solution (CCK-8 stock solution: DMEM = 1:50) was added to each well in the dark. Incubation continued for 4 hours. The absorbance of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the co-linking coefficient was calculated. The results are shown below. Figure 1 , Figure 1 The horizontal axis represents drug concentration in ng / ml, and the solvent is DMEM plus fetal bovine serum; the vertical axis represents survival rate.

[0033] The results showed that decanal at concentrations of 40 ng / ml, 60 ng / ml, and 80 ng / ml significantly promoted the proliferation and activity of bovine ovarian granulosa cells.

[0034] Example 2: Real-time cell growth monitoring technology to determine the effect of decanal on the proliferation of bovine ovarian granulosa cells

[0035] Experimental materials: Bovine ovaries were collected from the slaughterhouse, and decanal (analytical standard, GC=98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0036] Experimental Methods: Primary bovine ovarian granulosa cells were extracted and seeded at 5000 cells / well in 96-well plates. After cell adhesion, 200 μL of decanal (solvent: DMEM with fetal bovine serum) at different concentrations was added to each well. The cells were cultured in a Cytation 5 instrument for 48 h, with images taken every 6 h. The slope of cell growth was calculated based on cell confluence and analyzed. Results are shown below. Figure 2 .

[0037] The results showed that 50 ng / ml of decanal most significantly promoted the proliferation of bovine ovarian granulosa cells.

[0038] Example 3: Flow cytometry analysis of the effect of decanal treatment on the cell cycle of bovine ovarian granulosa cells

[0039] Experimental materials: Bovine ovaries were collected from the slaughterhouse; decanal (analytical standard, GC=98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and cell cycle and apoptosis detection kit (PI method) was purchased from Changchun Huayi Biotechnology Co., Ltd.

[0040] Experimental Methods: Primary bovine ovarian granulosa cells were extracted, and 3 million cells were inoculated into each large culture dish. After cell adhesion, 5 ml of 50 ng / ml decanal (solvent: DMEM with fetal bovine serum) was added, and the cells were treated with the drug for 24 hours. The cell culture medium was then discarded. The cells were digested with trypsin to prepare a single-cell suspension. The suspension was centrifuged at 1000 g for 3-5 min at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of pre-chilled PBS, centrifuged at 1000 g for 3-5 min at 4°C, and the supernatant was discarded, retaining the cell pellet. The cells were fixed by adding 1 mL of pre-chilled 70% ethanol to the collected cell pellet, gently pipetting to mix, and fixing at 4°C for 2 h or longer. (Note: Fixation at 4°C for 12-24 h yields better results.) The cells were washed, centrifuged at 1000 g for 3-5 min at 4°C, and the supernatant was discarded. Resuspend the cells in 1 mL of pre-chilled PBS, centrifuge at 1000 g for 3-5 min at 4°C, discard the supernatant, and retain the cell pellet. Prepare PI staining working solution (kit), making an appropriate amount of PI staining working solution according to the number of samples to be tested.

[0041] Table 1

[0042]

[0043] Note: PI staining working solution should be stored at 4°C protected from light for 24 hours. For staining, add 0.5 mL of PI staining working solution to each cell sample, gently resuspend the cell pellet, and incubate at 37°C protected from light for 30 min. For detection and analysis, use flow cytometry to detect red fluorescence at an excitation wavelength of 488 nm and assess light scattering. Analyze DNA content and light scattering using appropriate software.

[0044] The results showed that 50 μg / ml of decanal significantly promoted the number of S-phase cells in bovine ovarian granulosa cells. Figure 3 ).

[0045] Example 4: RT-qPCR detection of the effect of 50 ng / ml decanal treatment on proliferation-related genes in bovine ovarian granulosa cells

[0046] Experimental materials: Bovine ovaries were collected from the slaughterhouse, and decanal (analytical standard, GC=98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0047] Experimental methods: Primary bovine ovarian granulosa cells were extracted, and 3 million cells were inoculated into each large culture dish. After cell attachment, 5 ml of 50 ng / ml decanal (solvent: DMEM with fetal bovine serum) was added. After 48 h of drug treatment, the cells were collected with Trizol for RNA extraction, cDNA reverse transcription, and quantitative real-time PCR. The methods are as follows:

[0048] RNA extraction

[0049] (1) After the cells were washed three times with PBS, 1 mL of Trizol was added to each dish for lysis. The cells were then transferred to 1.5 mL RNAase-free centrifuge tubes and placed in an ice box for 10 min.

[0050] (2) After the cells are completely lysed, add 200 μL of chloroform to each centrifuge tube, mix and shake vigorously for 15 seconds, and place in an ice box to stand for 10 minutes.

[0051] (3) Centrifuge at 12,000 rpm for 15 min at 4℃ and take out the upper aqueous phase and mix it with an equal volume of isopropanol. Let it stand in an ice box for 10 min.

[0052] (4) Centrifuge at 4℃ and 12000rpm for 10min and discard the supernatant.

[0053] (5) Add 1 mL of 75% ethanol to each tube, centrifuge at 7500 rpm for 5 min at 4 °C, discard the supernatant, and dry the precipitate for 3 min.

[0054] (6) Add 10-20 μL of DEPC water to dissolve the precipitate until it is completely dissolved, and store it in a refrigerator at -80℃.

[0055] (7) The concentration and quality (OD260 / OD280) of RNA were detected using a nucleic acid spectrophotometer. A value between 1.8 and 2.0 indicates good purity. Reverse transcription (1) Remove the genome. The reaction system is shown in Table 1-3. Temperature: 42℃, time: 2 min.

[0056] Reverse transcription

[0057] (1) Remove the genome. The reaction system is shown in Table 2. Temperature: 42℃, time: 2min.

[0058] Table 2 Genome Removal Reaction System

[0059]

[0060] (2) Reverse transcription PCR, the reaction system is shown in Table 3, temperature: 37℃, time: 15min; temperature: 85℃, time: 5s.

[0061] Table 3 Reverse Transcription PCR Reaction System

[0062]

[0063] Real-time PCR detection

[0064] (1) All primers used in this experiment were designed and synthesized by Sangon Biotech Co., Ltd. The primer sequences are shown in the table.

[0065] Table 4 Primer Sequences for Quantitative Real-Time PCR

[0066]

[0067] (2) Prepare the PCR reaction system according to the SYBR® Premix Ex Taq™ II kit. The reaction conditions are shown in Table 5:

[0068] Table 5. Quantitative Real-Time PCR System

[0069]

[0070] (3) The reaction procedure for quantitative fluorescence is shown in Table 6, based on 2 -∆∆Ct The relative expression level of mRNA was calculated using this method.

[0071] Table 6. Quantitative Real-Time PCR Reaction Procedure

[0072]

[0073] The results are as follows Figure 4 Decanal significantly increased the expression levels of CDK1, CDC42, PCNA, cyclin A2, and cyclin D2, which are associated with granulocyte proliferation.

[0074] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of decanal in the preparation of formulations that enhance the fertility of female animals; wherein the female animal is a cow.

2. The application according to claim 1, characterized in that, The enhancement of female fertility includes promoting the proliferation of ovarian granulosa cells.

3. The application according to claim 2, characterized in that, The promotion of ovarian granulosa cell proliferation includes: Promotes the proliferation and activity of granulocytes; Increase the number of S-phase cells in granulocytes; And / or increase the expression levels of CDK1, CDC42, PCNA, cyclin A2 and / or cyclin D2 genes.

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