Method for improving superovulation efficiency of animals by using L-kynurenine

Abstract

The invention provides a method for improving animal superovulation efficiency by using L-kynurenine, and relates to the field of animal breeding. The method comprises the steps that L-kynurenine is injected into an animal, the injection dosage is 50 mg / kg of body weight, continuous injection is conducted for 7 days, and after injection is finished, superovulation treatment is conducted; according to the application, the characteristics that L-kynurenine can promote synthesis of glutathione in follicular granular cells and inhibit ferroptosis of the granular cells are found and utilized for the first time, so that follicular atresia is reduced, and follicular development of animals is improved; the injection of the L-kynurenine is combined with the superovulation treatment, so that the superovulation effect can be obviously improved, and more available ova can be obtained.

Description

Technical Field

[0001] The present invention relates to the field of animal husbandry and veterinary medicine, in particular to a method for improving the efficiency of animal superovulation. Technical Background

[0002] Superovulation and its supporting artificial insemination, in vitro fertilization and embryo transfer technologies are important technologies in livestock breeding and production. They fully tap the reproductive potential of high-quality female livestock in livestock production, reduce the waste of high-quality genetic resources, shorten the generation interval, and accelerate the speed of breed improvement and expansion of fine varieties.

[0003] The theory of superovulation is to recruit early preantral follicles through the stimulation of exogenous gonadotropins, causing them to grow rapidly and ovulate. Under natural conditions, the primordial follicles in the primordial follicle reservoir of the animal ovary are gradually activated, and a portion of them are recruited into the growth phase. Under the synergistic action of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), only a few dominant follicles are able to mature and participate in ovulation and fertilization, while the vast majority of the remaining subordinate follicles exit the developmental stage through programmed atresia. Therefore, the underlying logic of superovulation can also be understood as artificially increasing the level of gonadotropins on which follicle development depends in order to rescue non-dominant follicles that are about to atresize, while promoting their rapid growth, development, and ovulation, ultimately increasing the number of effective eggs released.

[0004] Superovulation currently has relatively mature protocols and hormonal regimens. Despite extensive research and significant progress in superovulation methods and influencing factors, the average number of viable eggs per superovulation cycle has barely increased since the method's invention. This bottleneck has necessitated a critical breakthrough in superovulation technology. Therefore, developing new technologies to increase superovulation efficiency is crucial.

[0005] L-kynurenine (CAS: 2922-83-0) is a metabolic intermediate in tryptophan. Over 95% of tryptophan in the body is processed into L-kynurenine by enzymes such as tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Currently, no method has been reported to utilize L-kynurenine to enhance superovulation efficiency. Summary of the Invention

[0006] In response to the long-standing problem that the number of eggs / embryos obtained by superovulation has not been significantly improved, the present application provides a method for improving the efficiency of superovulation by combining L-kynurenine treatment with superovulation.

[0007] Specifically, the above-mentioned object of the invention is achieved by the following method:

[0008] First, this application provides a method for improving the efficiency of superovulation in mammals using L-kynurenine, namely, injecting L-kynurenine into a mammal to improve the efficiency of superovulation, obtain more usable eggs / embryos, and improve the quality of eggs / embryos. The above-mentioned injection dose is preferably no more than 50 mg / kg animal body weight.

[0009] Furthermore, the L-kynurenine injection is prepared by completely dissolving 500 mg of L-kynurenine powder in a NaOH solution (preferably at a concentration of 1 mol / L), adjusting the pH of the solution to neutral with concentrated hydrochloric acid, and finally adding deionized water to a volume of 10 mL, so that the final concentration of L-kynurenine in the injection is 50 mg / mL.

[0010] Injection schedule and method: Inject mammals (e.g., mice, rabbits, etc.) intraperitoneally or intravenously with at least one (e.g., one, two, three, four, five, six, seven, etc.) injection of the aforementioned 50 mg / mL L-kynurenine injection, preferably once daily, into the peritoneal cavity or auricular vein. Each injection dose should not exceed 50 mg / kg (animal body weight). After all L-kynurenine injections are completed, begin conventional superovulation treatment three days later.

[0011] In one embodiment of the present application, the above-mentioned L-kynurenine injection refers to: injecting a 50 mg / mL L-kynurenine injection into the mammal once a day, the injection dose of L-kynurenine does not exceed 50 mg / kg (animal body weight), and the injection is carried out for a total of 7 days.

[0012] In one embodiment of the present application, the superovulation treatment refers to: after the L-kynurenine injection is completed, the mammal is injected with PMSG at an interval of 3 days, and then injected with hCG at an interval of 1 day.

[0013] Secondly, the present application provides an application of L-kynurenine in the preparation of a mammalian superovulation promoter.

[0014] Third, the present application provides a mammalian superovulation injection, which contains L-kynurenine at a concentration of 50 mg / mL; further, the promoter also includes other pharmaceutically acceptable excipients, diluents, etc.

[0015] In one embodiment of the present application, the injection is obtained by the following method: completely dissolving L-kynurenine powder in a NaOH solution (preferably at a concentration of 1 mol / L), then adjusting the pH of the solution to neutral, and finally adding deionized water until the final concentration of L-kynurenine in the injection is 50 mg / mL.

[0016] The present application has found for the first time that L-kynurenine can be detected in follicular fluid and blood, and its content in the follicular fluid of atretic follicles is significantly lower than that in healthy follicles, indicating that L-kynurenine may have a positive effect on follicular development. Further in vitro experiments have shown that L-kynurenine can promote GSH (glutathione) synthesis in granulosa cells (GC) of follicles, significantly inhibit the iron death of GC, significantly inhibit the iron death of granulosa cells, eliminate oxidative stress, and promote cell proliferation. Animal experiment results show that the number of preantral follicles in the ovaries of animals (such as mice and rabbits) can be increased, the number of atretic follicles can be reduced, thereby increasing the number of preantral follicles and improving superovulation efficiency. The present application further prepares L-kynurenine as a superovulation-promoting injection to be applied to improve superovulation in mammalian animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are paraffin sections of ovarian tissues of mice in the control group and experimental group of Example 2.

[0018] Figure 2 This is a schematic diagram of the statistical results of the number and atresia of follicles at various levels in the ovaries of mice in the control group and experimental group in Example 2.

[0019] Figure 3 This is a schematic diagram of the superovulation statistical results of Example 3. DETAILED DESCRIPTION

[0020] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0021] The reagents and materials used in the following examples are all commercially available products.

[0022] L-kynurenine solid powder was purchased from Aladdin Biochemical Technology Co., Ltd.

[0023] Example 1 Preparation of L-kynurenine injection

[0024] The injection is prepared by adding 500 mg of L-kynurenine in small amounts to a 1N (mol / L) NaOH solution several times, pipetting the solution after each addition to fully dissolve it. After complete dissolution, the pH of the solution is slowly adjusted to neutral (pH = 7) with 37% concentrated hydrochloric acid, and finally, deionized water is added to make the volume 10 mL, so that the final concentration of L-kynurenine in the injection is 50 mg / mL.

[0025] Example 2 L-kynurenine inhibits mouse follicle atresia experiment

[0026] 7- to 8-week-old female ICR mice (purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd.) were used as experimental animals. Mice were housed at 24-26°C and 50%-60% relative humidity with a 12-hour light-dark cycle. Food and water were available ad libitum, and bedding was changed weekly. Experimental treatments were performed after 3 days of adaptive feeding.

[0027] For this study, 7- to 8-week-old ICR female mice of similar size and good health were randomly divided into a control group (n = 45, injected with an equal volume of normal saline) and an L-kynurenine group (n = 45, injected with the L-kynurenine injection prepared in Example 1 at a dose of 50 mg / kg). The mice were weighed at 10:00 AM daily and intraperitoneal injections were performed.

[0028] The experimental groups were treated as follows: First, the mice were weighed, and then the tails of the mice were marked according to the groups. The drug solution was preheated to 37°C, and the dosage for each mouse was calculated as 50 mg / kg body weight. Finally, a sterile syringe was used to draw up the corresponding dose of L-kynurenine injection (Kyn) for injection. Before injection, the abdomen of the mouse was wiped with a 75% alcohol cotton ball. Grab the neck of the mouse with your left hand and turn its head down. Hold the syringe with your right hand and quickly insert the needle at a 45° angle into the lower abdomen on both sides, about 0.5 cm away from the midline. Slowly push the syringe piston to inject the drug solution into the abdominal cavity. After the injection is completed, rotate and pull out the needle, and gently press the injection site with an alcohol cotton ball to prevent drug leakage. During the entire operation, the principles of aseptic operation must be strictly followed to ensure the comfort and safety of the animals. The control group (Ctrl) was injected with an equal amount of normal saline, and the other treatment steps were the same as above.

[0029] 24 hours after the last administration, the mice were killed, the ovaries were removed, and the left ovary was fixed in paraformaldehyde for subsequent preparation of tissue paraffin sections and HE staining (the sections of the control group and the experimental group were as follows Figure 1 (as shown in a and b) to observe and count the number and atresia status of follicles at all levels in the ovary.

[0030] The results are as follows Figure 2 As shown, Figure 2 In the figure, a and b are the statistical results of the number of preantral follicles and atretic follicles, respectively. It can be seen that compared with the control group, the number of preantral follicles in the L-kynurenine treated group increased significantly, while the number of atretic follicles decreased significantly ( Figure 2 , P<0.001).

[0031] Example 3 Method for Improving Animal Superovulation Efficiency Using L-kynurenine, Superovulation Treatment of Mice

[0032] The selection, feeding and management of experimental animals and the injection of L-kynurenine in this example were carried out according to Example 2.

[0033] After the mice were continuously injected with L-kynurenine for 7 days (day -7 to day -1), superovulation treatment (conventional treatment method in the art) was performed on the 3rd day (i.e., 3 days after the L-kynurenine injection) starting from the next day (day 0). The specific treatment method is shown in Table 1 below.

[0034] In this example, the control group mice are the control group mice injected with physiological saline in Example 2.

[0035] Table 1 Methods for improving animal superovulation efficiency using L-kynurenine

[0036] date deal with -7 days to -1 day, 10:00 am Intraperitoneal injection of L-kynurenine 50 mg / kg 0-2 days - 3 days, 5 p.m. Intraperitoneal injection of PMSG (pregnant mare serum gonadotropin) 5 IU / animal 5 days, 3 p.m. Intraperitoneal injection of hCG (human chorionic gonadotropin) 5 IU / head 6 days Fallopian tube oocyte retrieval

[0037] 15-18 hours after superovulation, the mice were killed, the abdominal cavity was cut open to expose the uterus, and the oviduct between the cervix and the ovary was cut. In a culture dish filled with physiological saline, the enlarged ampulla of the oviduct was found under a microscope and punctured with a needle to release the oocytes. The number of oocytes released was counted. Figure 3 As shown in the figure, compared with the control group injected with normal saline, the number of oocytes obtained in the experimental group injected with L-kynurenine was significantly increased (29.7 vs 45.4, P = 0.014): Normal superovulation treatment can obtain about 30 eggs, while injection of L-kynurenine can increase the efficiency of superovulation to about 45 eggs, an increase of 15%.

[0038] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for improving the efficiency of superovulation in animals by utilizing L-kynurenine, characterized in that: The method is to inject L-kynurenine injection into animals, and start superovulation treatment 3 days after the injection to improve the superovulation efficiency of animals.

2. The method for improving the efficiency of superovulation of animals by utilizing L-kynurenine according to claim 1, characterized in that: The injection refers to at least one injection of L-kynurenine injection, and the injection dose of L-kynurenine each time does not exceed 50 mg / kg animal body weight.

3. The method for improving the efficiency of superovulation of animals by utilizing L-kynurenine according to claim 1, characterized in that: The injection method includes intravenous injection or intraperitoneal injection.

4. The method for improving the efficiency of superovulation of animals by utilizing L-kynurenine according to claim 1, characterized in that: The L-kynurenine injection is prepared by completely dissolving L-kynurenine powder in a NaOH solution, adjusting the pH of the solution to neutral, and adding deionized water until the final concentration of L-kynurenine is 50 mg / mL, thereby obtaining the L-kynurenine injection.

5. The method for improving the efficiency of superovulation of animals by utilizing L-kynurenine according to claim 1, characterized in that: The superovulation treatment refers to injecting PMSG into the animals and then injecting hCG one day later.

6. The method for improving the efficiency of superovulation of animals by utilizing L-kynurenine according to claim 1, characterized in that: The animals include mice and rabbits.

7. The method for improving the efficiency of superovulation of animals using L-kynurenine according to claim 2, characterized in that: The injection refers to injecting L-kynurenine injection once a day for a total of 7 days.

8. Application of L-kynurenine in the preparation of mammalian superovulation promoter.

9. A mammalian superovulation injection, comprising L-kynurenine at a concentration of 50 mg / mL.

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