A traditional Chinese medicine composition for improving reproductive performance, a preparation method and application thereof

Abstract

The application discloses a traditional Chinese medicine composition for improving reproductive performance, a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicine preparation and reproductive regulation. The traditional Chinese medicine composition comprises the following components in mass fractions: 14-16 parts of radix codonopsis, 14-16 parts of radix astragali, 8-12 parts of rhizoma atractylodis macrocephalae, 18-22 parts of poria cocos, 8-12 parts of magnolia officinalis, 8-12 parts of radix ophiopogonis, 5-7 parts of ramulus cinnamomi, 5-7 parts of paeonia lactiflora, 2-4 parts of cassia bark, 18-22 parts of medlar and 8-12 parts of honey-fried licorice root. The above-mentioned medicinal materials are proportioned, soaked, decocted twice, filtered, concentrated and prepared into water decoction containing 1 g / mL of crude drug. The traditional Chinese medicine composition has the effects of invigorating the spleen and replenishing qi, nourishing yin and blood, warming yang and dredging collaterals, regulating qi and blood, tonifying liver and kidney, improving oxidative stress state and regulating reproductive function, and can be used for preparing medicines or feed additive preparations for improving reproductive performance, improving reproductive function decline and improving gamete quality.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparations and reproductive regulation technology, and relates to a traditional Chinese medicine composition for improving reproductive performance, its preparation method and application. In particular, it relates to the application of the traditional Chinese medicine composition in improving pregnancy rate, litter size, follicle development, sperm quality, reproductive hormone levels and oxidative stress in mammals. Background Technology

[0002] Reproductive performance is an important indicator for measuring the productive efficiency, population maintenance capacity, and reproductive quality of mammals, particularly laboratory animals. For humans and other mammals, reproductive performance is influenced by multiple factors, including reproductive endocrine status, ovarian reserve, oocyte quality, sperm count and motility, metabolic level, and oxidative stress. With age or a decline in bodily function, females often experience decreased ovarian reserve, reduced oocyte quality, and lower pregnancy rates, while males may exhibit reduced sperm count, decreased sperm motility, increased sperm abnormality rate, and reproductive endocrine dysfunction, thus affecting overall reproductive performance.

[0003] Existing methods for improving reproductive performance mainly include optimizing nutrient supply, improving the environment and reproductive management, applying assisted reproductive technologies, and using exogenous hormone intervention. Although methods such as exogenous gonadotropins, hormone-induced ovulation, or superovulation can increase the number of ovulations or improve short-term reproductive results to some extent, they usually have drawbacks such as being complex to operate, relying on professional skills, being costly, and potentially interfering with the body's endocrine homeostasis. Especially for older individuals or individuals with declining reproductive function, simply relying on exogenous hormone stimulation is often insufficient to improve ovarian function, the reproductive organ microenvironment, and reproductive system homeostasis as a whole. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a traditional Chinese medicine composition for improving reproductive performance, aiming to provide a safe, stable and easy-to-apply traditional Chinese medicine intervention scheme for improving the reproductive performance of mammals, alleviating reproductive function decline, and improving gamete quality.

[0005] Another object of the present invention is to provide a method for preparing the traditional Chinese medicine composition.

[0006] A third objective of this invention is to provide the application of this traditional Chinese medicine composition.

[0007] The technical solution of the present invention is: a traditional Chinese medicine composition for improving reproductive performance, the traditional Chinese medicine composition comprising the following components in parts by weight: Codonopsis pilosula 14-16 parts, Astragalus membranaceus 14-16 parts, Atractylodes macrocephala 8-12 parts, Poria cocos 18-22 parts, Magnolia officinalis 8-12 parts, Ophiopogon japonicus 8-12 parts, Cinnamomum cassia 5-7 parts, Paeonia lactiflora 5-7 parts, Cinnamomum cassia 2-4 parts, Lycium barbarum 18-22 parts, and Glycyrrhiza uralensis (processed) 8-12 parts.

[0008] Furthermore, the traditional Chinese medicine composition comprises the following components in parts by weight: 15 parts Codonopsis pilosula, 15 parts Astragalus membranaceus, 10 parts Atractylodes macrocephala, 20 parts Poria cocos, 10 parts Magnolia officinalis, 10 parts Ophiopogon japonicus, 6 parts Cinnamomum cassia, 6 parts Paeonia lactiflora, 3 parts Cinnamomum cassia, 20 parts Lycium barbarum, and 10 parts Glycyrrhiza uralensis (processed).

[0009] Furthermore, the preparation method of the traditional Chinese medicine composition for improving reproductive performance includes the following steps: Step (1) Soak the raw materials to be decocted in 2 to 4 times (preferably 3 times) volume of distilled water for 1 to 2 hours (preferably 1 hour). Step (2) After soaking, bring the soaked raw materials to a boil over high heat. After boiling, reduce to a simmer and continue simmering for 1.5 to 2.5 hours (preferred: 2 hours). After simmering, filter the dregs through an 80-mesh sieve and collect the liquid. The filtered dregs will be simmered again. In step (3), add 2 to 4 times (preferably 3 times) of distilled water to the filtered residue, bring to a boil over high heat, and then simmer over low heat for 1.5 to 2.5 hours (preferably 2 hours). After simmering, filter the residue through an 80-mesh sieve and discard it, then collect the decoction. Combine the two decoctions and simmer over low heat to concentrate the decoction to the same volume as the raw materials to make the final decoction.

[0010] Furthermore, the application of the aforementioned traditional Chinese medicine composition in the preparation of materials that improve reproductive performance.

[0011] Furthermore, the application includes its use in the preparation of drugs to improve pregnancy rates and litter sizes in young and late-reproductive-age ICR female mice.

[0012] Furthermore, the application includes its use in the preparation of drugs to enhance in vitro fertilization after overovulation in young and late-reproductive-age ICR female mice.

[0013] Furthermore, the application includes its use in the preparation of drugs that enhance follicle development in young and late-reproductive-age ICR female mice.

[0014] Furthermore, the application includes its use in the preparation of drugs to improve sperm quality in young and late-reproductive-age ICR male rats.

[0015] Furthermore, the application includes its use in the preparation of drugs that improve the morphology of epididymal tissue in young and late-fertile ICR male rats.

[0016] Furthermore, the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs for improving the levels of reproductive-related hormones in the peripheral blood of young and late-reproductive-age ICR female and male mice.

[0017] Furthermore, the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs to improve peripheral blood oxidative stress indicators in young and late-reproductive-age ICR female and male mice.

[0018] A traditional Chinese medicine composition for improving reproductive performance is prepared by soaking 1000 g of the composition in three times its volume of distilled water for 1.5 h after boiling. The mixture is then simmered over low heat for 2 h, and the liquid is filtered through an 80-mesh sieve. Three times its volume of distilled water is added to the residue, and the mixture is simmered again for 2 h, then filtered through an 80-mesh sieve. The two decoctions are combined and concentrated to 1000 mL over low heat. After filtration, a 1 g / mL aqueous decoction is obtained. The administration method is drinking water, and the dosage is 0.02 g / kg.

[0019] The herbal composition for improving reproductive performance consists of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum and 10 g of Glycyrrhiza uralensis (processed). The combination of Codonopsis pilosula, Astragalus membranaceus, Atractylodes macrocephala, and Poria cocos has the effects of strengthening the spleen and replenishing qi, consolidating qi and strengthening the body's foundation, and improving the body's metabolism and nutritional status, which can provide basic support for improving reproductive function; Magnolia officinalis can promote qi circulation and eliminate dampness, regulate qi mechanism, and help improve the body's digestion, absorption and metabolism; Ophiopogon japonicus, Paeonia lactiflora, and Lycium barbarum have the effects of nourishing yin and generating fluids, nourishing blood and softening the liver, tonifying the liver and kidneys, and benefiting essence and nourishing blood, which can improve age-related functional decline and help improve ovarian function, sperm quality and antioxidant capacity; Cinnamomum cassia and Cinnamomum cassia have the effects of warming yang and unblocking meridians, and warming and tonifying kidney yang, which can promote the circulation of qi and blood and improve reproductive function decline; Glycyrrhiza uralensis has the effects of tonifying the spleen and replenishing qi, and harmonizing the effects of various drugs, which can enhance the overall coordination and comprehensive regulatory effect of the formula.

[0020] Traditional Chinese medicine (TCM) and its compound preparations are characterized by multiple components, multiple targets, and multiple regulatory mechanisms, offering potential advantages in improving bodily functions, promoting tissue repair, regulating immunity, and delaying aging. TCM theory holds that the kidneys store essence and govern reproduction, while the spleen is the source of qi and blood production. The essence of food and drink acquired after birth nourishes the innate kidney essence. Deficiency of the spleen and kidneys, and insufficiency of qi and blood, can lead to malnourishment of the Chong and Ren meridians, insufficient nourishment of the uterus, or inadequate filling of the seminal vesicles, thereby affecting the establishment of pregnancy, embryonic survival, follicle development, and spermatogenesis. Therefore, constructing TCM formulas from the perspectives of strengthening the spleen and replenishing qi, warming yang and unblocking the meridians, nourishing yin and blood, and tonifying the liver and kidneys may be an effective strategy for improving reproductive performance and alleviating reproductive dysfunction in mammals.

[0021] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: 1. The composition of the present invention comprehensively invigorates the spleen and replenishes qi, nourishes yin and blood, warms yang and unblocks the meridians, tonifies the liver and kidneys, and harmonizes qi and blood, thus improving the overall reproductive function of male and female mammals; 2. The traditional Chinese medicine composition of the present invention can significantly improve the reproductive performance of female mammals. Animal experiments show that after treatment with the traditional Chinese medicine composition, the pregnancy rate and litter size of ICR female mice increased, the number of fertilized eggs obtained by in vitro fertilization after superovulation increased, and the development of follicles and corpus luteum at all stages in the ovary was improved; 3. The traditional Chinese medicine composition of the present invention can improve the reproductive function of male mammals. Animal experiments show that after treatment with the traditional Chinese medicine composition, the overall sperm motility of ICR male mice improved, the epididymal seminiferous tubules became more morphologically complete, the sperm density in the lumen increased, and the serum testosterone level increased; 4. The traditional Chinese medicine composition of the present invention can regulate reproductive endocrine and oxidative stress states. Animal experiments showed that after treatment with the traditional Chinese medicine composition, the levels of estradiol in female mice increased and the levels of follicle-stimulating hormone decreased, while the levels of testosterone in male mice increased. At the same time, the levels of MDA in peripheral blood decreased and the activities of SOD and GSH-Px increased, suggesting that it can participate in improving reproductive performance by improving oxidative stress. Attached Figure Description

[0022] Figure 1 This is a comparison chart of pregnancy rate and litter size among different experimental groups of young ICR female mice in this invention embodiment; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P < 0.01, * P < 0.05; Figure 2 This is a schematic diagram showing the number of fertilized eggs after in vitro fertilization in each experimental group of young ICR female mice in this embodiment of the invention; Figure 3 This is an observation of stained sections of ovarian tissue and statistical charts of follicles at various stages in different experimental groups of young ICR female mice in this invention embodiment; Note: Data are the mean ± standard deviation of 3 parallel samples; compared with the Con group, *** P < 0.001, ** P <0.01, * P < 0.05; Figure 4 This is a comparison chart of peripheral blood reproductive-related hormone levels in different experimental groups of young ICR female mice in this invention embodiment; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P < 0.01, * P < 0.05; Figure 5 This is a schematic diagram of peripheral blood oxidative stress-related indicators in each experimental group of young ICR female mice in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P< 0.01, * P < 0.05; Figure 6 and Figure 7 This is a schematic diagram of sperm quality in each experimental group of young ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P < 0.01, * P < 0.05; Figure 8 This is a schematic diagram of epididymal tissue staining sections observed in various experimental groups of young ICR male rats in this invention embodiment; Figure 9 This is a schematic diagram of the peripheral blood reproductive-related hormone levels in each experimental group of young ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P < 0.01, * P < 0.05; Figure 10 This is a schematic diagram of peripheral blood oxidative stress-related indicators in each experimental group of young ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P < 0.01, * P < 0.05; Figure 11 This is a schematic diagram showing the pregnancy rate and litter size of late-reproductive-age ICR female mice in each experimental group in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P < 0.01, * P < 0.05; Figure 12 This is a schematic diagram showing the number of fertilized eggs after in vitro fertilization in each experimental group of late-fertility ICR female mice in this embodiment of the invention. Figure 13 This is a schematic diagram illustrating the observation of stained sections of ovarian tissue and statistical analysis of follicles at various stages in late-reproductive-age ICR female mice in various experimental groups according to embodiments of the present invention; Note: Data are the mean ± standard deviation of 3 parallel samples; compared with the Con group, *** P <0.001,** P < 0.01; Figure 14 This is a schematic diagram of peripheral blood reproductive-related hormone levels in late-reproductive-age ICR female mice in each experimental group according to embodiments of the present invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, **P < 0.01, * P < 0.05; Figure 15 This is a schematic diagram of peripheral blood oxidative stress-related indicators in late-fertility ICR female mice in various experimental groups according to embodiments of the present invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P < 0.01, * P < 0.05; Figure 16 and Figure 17 This is a schematic diagram of sperm quality in each experimental group of late-fertility ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, ** P < 0.01, * P < 0.05; Figure 18 This is a schematic diagram of epididymal tissue staining sections observed in various experimental groups of late-fertility ICR male rats in this embodiment of the invention; Figure 19 This is a schematic diagram of peripheral blood reproductive-related hormone levels in late-fertility ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P < 0.01, * P < 0.05; Figure 20 This is a schematic diagram of peripheral blood oxidative stress-related indicators in late-fertility ICR male rats in this embodiment of the invention; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P < 0.01, * P < 0.05. Detailed Implementation

[0023] The specific technical solutions of the present invention will be further described in detail below with reference to specific examples.

[0024] The traditional Chinese medicine preparation for improving reproductive performance provided in this embodiment of the invention is composed of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum and 10 g of Glycyrrhiza uralensis.

[0025] The traditional Chinese medicine composition of this invention has the effects of invigorating the spleen and replenishing qi, nourishing yin and blood, warming yang and unblocking collaterals, tonifying the liver and kidneys and regulating qi and blood, and can improve the reproductive function of male and female mammals as a whole.

[0026] Example 1: Effects of different traditional Chinese medicine compositions on improving the reproductive performance of young female ICR rats 1. Method 1.1 Laboratory Animals and Grouping Processing 1.1.1 Effects of Traditional Chinese Medicine Formula on Pregnancy Rate and Litter Size in Young ICR Female Rats Ninety-six young female ICR mice were randomly divided into four groups of 24 mice each: a blank control group (Con), a one-week treatment group (TCSF-1W), a two-week treatment group (TCSF-2W), and a three-week treatment group (TCSF-3W). The Con group received 240 mL of autoclaved water daily, while the TCSF-3W group received 12 mL of a traditional Chinese medicine decoction and 228 mL of autoclaved water daily starting from day 1 of the experiment. The TCSF-2W group received the same drug solution from day 8 of the experiment, and the TCSF-1W group received the same drug solution from day 15 of the experiment. Freshly prepared Chinese herbal decoction and autoclaved water were changed daily in each group. On day 22 of the experiment, a total of 24 female ICR mice from each group were divided into 12 cages at a density of 2 mice per cage. According to the principle of female to male ratio of 2:1, 48 male mice were randomly assigned to each cage for mating. After 3 days of mating, the female mice from each group were put together for further observation of pregnancy rate and litter size. 1.1.2 Effects of Traditional Chinese Medicine Formula on Reproductive Function of Young ICR Female Rats Eighty young female ICR mice were randomly divided into four groups of 20 mice each: Con group, TCSF-1W group, TCSF-2W group, and TCSF-3W group. The Con group received 200 mL of autoclaved water daily. The TCSF-3W group received a mixture of 10 mL of herbal decoction and 190 mL of autoclaved water daily from day 1 of the experiment. The TCSF-2W group received the same mixture from day 8, and the TCSF-1W group received the same mixture from day 15. Freshly prepared herbal decoction and autoclaved water were provided daily for each group. On day 22, each mouse was injected with 0.1 mL of 50 IU / mL pregnant horse serum gonadotropin (PMSG) and 0.1 mL of 50 IU / mL human chorionic gonadotropin (hCG). mL, to synchronize estrus and superovulation treatment; after intraperitoneal injection of Zoltil® 50 anesthesia, blood and ovaries were collected from each group of female mice; the ovaries of 10 female mice were removed and placed directly in fixative for subsequent HE staining and sectioning observation; the ovaries of the other 10 female mice were separated from the oviduct dilatation under a stereomicroscope, and oocytes were picked for in vitro fertilization; after the samples were collected, all experimental animals were uniformly anesthetized and euthanized by cervical dislocation, and then disposed of in a harmless manner; 1.2 Composition of Traditional Chinese Medicine Compounds and Preparation of Decoction The traditional Chinese medicine preparation for improving reproductive performance provided in this invention consists of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum, and 10 g of Glycyrrhiza uralensis (processed). The traditional Chinese medicine composition of this invention has the effects of strengthening the spleen and replenishing qi, nourishing yin and blood, warming yang and unblocking collaterals, tonifying the liver and kidneys, and regulating qi and blood, which can improve the reproductive function of male and female mammals as a whole. Take 1000 g of the herbal composition according to the specified ratio, add 3 times its volume of distilled water and soak for 1.5 h; after boiling, simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve; add 3 times its volume of distilled water to the dregs, continue to simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve. Combine the two decoctions and concentrate them over low heat to 1000 mL, then filter to obtain a 1 g / mL aqueous decoction. The administration method is drinking water, and the dosage is 0.02 g / kg. 1.3. Statistics on pregnancy rate and litter size in young female ICR mice The number of pregnant female mice and the number of offspring in each group were recorded, the pregnancy rate was calculated, and the average number of offspring in each group was statistically analyzed to evaluate the effect of the traditional Chinese medicine formula on the reproductive performance of young ICR female mice. 1.4 Statistical observation of fertilized eggs from young ICR female rats After drug administration, young ICR female mice underwent superovulation treatment, and ovarian and oviduct tissues were collected. The ovaries were washed in 37°C pre-warmed PBS to remove adipose tissue, and then transferred to 37°C pre-warmed M2 processing solution. The ampulla of the oviduct was cut open under a stereomicroscope to release the cumulus-oocyte complex. At the same time, epididymal sperm from male mice was collected, capacitated at 37°C and 5% CO2 for 10-15 min, and then co-incubated with the cumulus-oocyte complex in fertilization fluid droplets for 6 h. After fertilization, the cumulus cells were removed with M2 processing solution containing hyaluronidase, and the mice were washed 2-3 times with fresh M2 solution. Finally, the number of fertilized eggs was observed and counted under an inverted microscope. 1.5. Observation of stained sections of ovarian tissue and statistics of follicles at each stage in young ICR female rats Ovarian tissue was collected from young ICR female mice in each group, fixed, and trimmed into tissue blocks of approximately 5 × 5 × 2 mm. The blocks were rinsed in running water overnight. Subsequently, the tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then cut into 4 μm thick paraffin sections. After dewaxing and rehydration with graded ethanol, the sections were stained with hematoxylin and eosin and mounted with neutral resin. After staining, the morphology of the ovarian tissue was observed under an upright microscope, and the number of primordial follicles, primary follicles, secondary follicles, mature follicles, and corpora lutea was counted to evaluate the effect of the traditional Chinese medicine formula on follicular development in young ICR female mice. 1.6 Detection of peripheral blood reproductive hormone levels in young female ICR rats Peripheral blood was collected from young female ICR mice in each group. After centrifugation at 3000 r / min, the supernatant serum was collected. Following the ELISA kit instructions, standards or serum samples were added to pre-coated 96-well plates, followed by the addition of HRP-labeled detection antibody. The plates were incubated at 37°C for 60 min. After washing, substrate solution was added for color development, and the plates were incubated at 30°C in the dark for 15 min. The reaction was then terminated with stop solution, and the OD value was read at 450 nm. The levels of follicle-stimulating hormone (FSH) and estradiol (E2) in the samples were calculated based on the standard curve. 1.7 Detection of peripheral blood oxidative stress-related indicators in young female ICR rats Peripheral blood was collected from young female ICR rats and centrifuged to obtain serum. Oxidative stress-related indicators were detected according to the corresponding kit instructions. Serum samples were used to determine superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, and malondialdehyde (MDA) content. SOD was measured at 450 nm after incubation at 37℃; GSH-PX was measured at 412 nm after reaction at room temperature; and MDA was measured at 532 nm after reaction in a 95℃ water bath, cooling, and centrifugation. The levels of each indicator were calculated according to the kit instructions to evaluate the effect of the traditional Chinese medicine formula on the oxidative stress status of young female ICR rats. 1.8 Data Analysis Statistical analysis was performed using IBM SPSS Statistics 17.0 software, and graphs were created using Graphpad Prism 9.5.1. All data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons between groups. The criterion for statistical significance was... P <0.05.

[0027] 2. Experimental Results 2.1 Pregnancy rate and litter size in each experimental group of young ICR female mice like Figure 1 As shown, compared with the Con group, the pregnancy rate of mice in the TCSF-1W group was significantly higher. P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant improvement ( P < 0.01); Compared with the Con group, the number of pups in the TCSF-1W group was not significantly different, while the number in the TCSF-2W group was significantly higher ( P < 0.05), the TCSF-3W group showed a highly significant increase ( P < 0.01); 2.2 Number of fertilized eggs after in vitro fertilization in each experimental group of young ICR female mice like Figure 2 As shown, the number of fertilized eggs in female mice in the Con group was 8, the number of fertilized eggs in female mice in the TCSF-1W group was 10, the number of fertilized eggs in female mice in the TCSF-2W group was 12, and the number of fertilized eggs in female mice in the TCSF-3W group was 15; the number of fertilized eggs in female mice gradually increased with the duration of drug administration. 2.3 Observation of stained sections of ovarian tissue and statistics of follicles at each stage in different experimental groups of young ICR female rats like Figure 3As shown in Table 1, compared with the Con group, the number of corpora lutea in mice in both the TCSF-1W and TCSF-3W groups was significantly increased. P < 0.01), there was no significant difference in the TCSF-2W group; compared with the Con group, the number of primordial follicles in the TCSF-1W group was significantly increased ( P < 0.05), the TCSF-2W group showed a highly significant increase ( P < 0.001), the TCSF-3W group showed a highly significant increase ( P < 0.01); Compared with the Con group, the number of primary follicles in the TCSF-1W group was not significantly different, while the number in the TCSF-2W group was significantly increased ( P < 0.01), the TCSF-3W group showed a highly significant increase ( P < 0.001); Compared with the Con group, the number of secondary follicles in the TCSF-1W group mice was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P < 0.01); Compared with the Con group, the number of mature follicles in the TCSF-1W and TCSF-2W groups was not significantly different, while the number in the TCSF-3W group was significantly increased ( P < 0.01); Table 1. Number of follicles and corpora lutea in ovarian sections of female rats from different groups of young ICR male rats. ; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P <0.01, * P < 0.05;

[0028] 2.4 Peripheral blood levels of reproductive hormones in young female ICR rats in different experimental groups like Figure 4 As shown, compared with the Con group, the FSH levels in the TCSF-1W group mice were not significantly different, while those in the TCSF-2W group were significantly lower. P < 0.05), the TCSF-3W group showed a highly significant decrease ( P < 0.01); Compared with the Con group, the E2 content of mice in the TCSF-1W group was significantly increased (P < 0.05), and the levels in the TCSF-2W and TCSF-3W groups were extremely significantly increased (P < 0.01). P < 0.01); 2.5 Peripheral blood oxidative stress-related indicators in different experimental groups of young ICR female rats like Figure 5As shown, compared with the Con group, the SOD content of mice in the TCSF-1W group was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P < 0.01); Compared with the Con group, the GSH-PX content in mice in both the TCSF-1W and TCSF-2W groups was significantly increased ( P < 0.05), the TCSF-3W group showed a highly significant increase ( P < 0.01); Compared with the Con group, the MDA content of mice in both the TCSF-1W and TCSF-2W groups was significantly reduced ( P < 0.05), the TCSF-3W group showed a highly significant decrease ( P < 0.01).

[0029] 3. Summary The traditional Chinese medicine formula significantly improved the pregnancy rate, litter size, and number of in vitro fertilized eggs in young ICR female mice, promoted follicle development and corpus luteum formation, and improved peripheral blood reproductive hormone levels and oxidative stress. Its effects increased with prolonged administration time, suggesting that the formula can improve the reproductive performance of young ICR female mice by regulating reproductive endocrine function, enhancing antioxidant capacity, and improving ovarian function.

[0030] Example 2: Effects of different traditional Chinese medicine compositions on improving the reproductive performance of young ICR male rats 1. Method 1.1 Laboratory Animals and Grouping Processing Forty young male ICR rats were randomly divided into four groups of 10 each: Con group, TCSF-1W group, TCSF-2W group, and TCSF-3W group. The Con group received 100 mL of autoclaved water daily, while the TCSF-3W group received 5 mL of a traditional Chinese medicine decoction daily starting from day 1. The mixed drinking water was prepared with mL of autoclaved water. The TCSF-2W group received the same drug solution from day 8 of the experiment, while the TCSF-1W group received the same drug solution from day 15 of the experiment. Freshly prepared Chinese herbal decoction and autoclaved water were changed daily in each group. On day 22 of the experiment, male mice in each group were anesthetized by intraperitoneal injection of Zoltil® 50, and blood and epididymis were collected. The epididymis of 5 male mice were used for sperm motility testing, and the epididymis of the other 5 male mice were removed and immediately placed in fixative for subsequent HE staining and sectioning observation. After the samples were collected, all experimental animals were euthanized by cervical dislocation under anesthesia and disposed of in a harmless manner. 1.2 Composition of Traditional Chinese Medicine Compounds and Preparation of Decoction The traditional Chinese medicine preparation for improving reproductive performance provided in this invention consists of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum, and 10 g of Glycyrrhiza uralensis (processed). The traditional Chinese medicine composition of this invention has the effects of strengthening the spleen and replenishing qi, nourishing yin and blood, warming yang and unblocking collaterals, tonifying the liver and kidneys, and regulating qi and blood, which can improve the reproductive function of male and female mammals as a whole. Take 1000 g of the herbal composition according to the specified ratio, add 3 times the volume of distilled water and soak for 1.5 h; after boiling, simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve; add 3 times the volume of distilled water to the dregs, continue to simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve. Combine the two decoctions and concentrate them over low heat to 1000 mL, filter and prepare a 1 g / mL decoction; administer by drinking water, with a dosage of 0.02 g / kg; 1.3. Sperm motility detection in young ICR male rats The collected epididymis was placed in PBS preheated to 37°C. The epididymis was carefully removed, and a cross-shaped incision was made in it with scissors. The epididymis was then immersed in capacitation solution preheated to 37°C to allow sperm to swim out naturally. After 3 minutes, 5 μL of semen sample was collected and added to a Markler sperm counting chamber. Three clear fields of view were randomly selected. Sperm motility and various movement data were analyzed using software. Incorrectly labeled sperm were manually corrected, and the results were recorded. The parameters detected included curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), total motor sperm rate (TM), linearity index (LIN), and straightness index (STRAIL). STR) and vibration index (Wobble, WOB); 1.4 Observation of stained sections of epididymal tissue from young ICR male rats Epididymal tissue was collected from young male ICR rats in each group. After fixation, the tissue was trimmed into approximately 5 × 5 × 2 mm blocks and rinsed overnight in running water. Subsequently, the tissue was dehydrated with a series of ethanol gradients, cleared with xylene, embedded in paraffin, and sectioned into 4 μm thick paraffin sections. After dewaxing and rehydration with a series of ethanol gradients, the sections were stained with hematoxylin and eosin and mounted with neutral resin. After staining, the structure of the vas deferens and sperm density in the epididymis of male rats were observed under an upright microscope to evaluate the effect of the traditional Chinese medicine formula on the development of the epididymis in young male ICR rats. 1.5 Detection of peripheral blood reproductive-related hormone levels in young ICR male rats Peripheral blood was collected from each group of young male ICR rats. After centrifugation at 3000 r / min, the supernatant serum was collected. Following the ELISA kit instructions, the standard or serum sample was added to a pre-coated 96-well plate, followed by the addition of HRP-labeled detection antibody. The plate was incubated at 37°C for 60 min. After washing, substrate solution was added for color development, and the plate was incubated at 30°C in the dark for 15 min. The reaction was then terminated by adding stop solution, and the OD value was read at 450 nm. The testosterone (T) level in the peripheral blood of young male ICR rats was calculated based on the standard curve. 1.6 Detection of peripheral blood oxidative stress-related indicators in young male ICR rats Peripheral blood was collected from young male ICR rats and centrifuged to obtain serum. Oxidative stress-related indicators were detected according to the corresponding kit instructions. Serum samples were used to determine superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, and malondialdehyde (MDA) content. SOD was measured at 450 nm after incubation at 37℃; GSH-PX was measured at 412 nm after reaction at room temperature; and MDA was measured at 532 nm after reaction in a 95℃ water bath, cooling, and centrifugation. The levels of each indicator in the peripheral blood of young male ICR rats were calculated according to the kit instructions to evaluate the effect of the traditional Chinese medicine formula on the oxidative stress status of young male ICR rats. 1.7 Data Analysis Statistical analysis was performed using IBM SPSS Statistics 17.0 software, and graphs were created using Graphpad Prism 9.5.1. All data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons between groups. The criterion for statistical significance was... P <0.05.

[0031] 2. Experimental Results 2.1 Sperm quality in each experimental group of young ICR male rats like Figure 6 As shown, compared with the Con group, the VCL of mice in the TCSF-1W group was significantly increased ( P < 0.05), the TCSF-2W group showed a highly significant increase ( P < 0.01), the TCSF-3W group showed a highly significant increase ( P < 0.001); Compared with the Con group, the VSL of mice in both the TCSF-1W and TCSF-2W groups was significantly increased ( P < 0.05), the TCSF-3W group showed a highly significant increase ( P < 0.01); Compared with the Con group, the VAP of mice in the TCSF-1W group was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P <0.01); Compared with the Con group, there was no significant difference in ALH levels in the TCSF-1W and TCSF-2W groups, but the TCSF-3W group showed a significant increase ( P < 0.05); Compared with the Con group, there was no significant difference in BCF in the TCSF-1W and TCSF-2W groups, but the TCSF-3W group showed a significant increase ( P < 0.05); Compared with the Con group, there was no significant difference in TM in the TCSF-1W group, while it was significantly increased in the TCSF-2W group ( P < 0.05), the TCSF-3W group showed a highly significant increase ( P < 0.01); Compared with the Con group, LIN levels in mice in the TCSF-1W, TCSF-2W, and TCSF-3W groups were all decreased, but there was no statistically significant difference; Compared with the Con group, STR levels in mice in the TCSF-1W, TCSF-2W, and TCSF-3W groups were not significantly different; Compared with the Con group, WOB levels in mice in the TCSF-1W group were not significantly different, while WOB levels in the TCSF-2W and TCSF-3W groups were significantly decreased ( P < 0.05); like Figure 7 As shown, with the extension of drug administration time, the number of sperm and motility both showed an increasing trend, while the abnormality rate showed a decreasing trend. Among them, the improvement effect was most obvious in male mice in the TCSF-3W group. 2.2 Observation of epididymal tissue sections stained in different experimental groups of young ICR male rats like Figure 8 As shown, compared with the Con group, the epididymal tubules of young male rats in the TCSF-1W, TCSF-2W, and TCSF-3W groups were more robust, and the sperm density increased. 2.3 Peripheral blood levels of reproductive hormones in different experimental groups of young ICR male rats like Figure 9 As shown, compared with the Con group, the T content in the TCSF-1W group mice was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P < 0.01); 2.4 Peripheral blood oxidative stress-related indicators in different experimental groups of young ICR male rats like Figure 10 As shown, compared with the Con group, the SOD content of mice in the TCSF-1W group was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P < 0.01); Compared with the Con group, the GSH-PX content in the TCSF-1W group mice was significantly increased ( P < 0.05), both the TCSF-2W and TCSF-3W groups showed extremely significant increases ( P < 0.01); Compared with the Con group, the MDA content of mice in both the TCSF-1W and TCSF-2W groups was significantly reduced ( P < 0.05), the TCSF-3W group showed a highly significant decrease ( P < 0.01).

[0032] 3. Summary The traditional Chinese medicine formula can improve sperm motility and sperm density in young ICR male rats, improve epididymal tissue morphology, increase testosterone levels, and enhance antioxidant capacity and reduce oxidative damage; its improvement effect increases with the duration of administration.

[0033] Example 3: Effects of different traditional Chinese medicine compositions on improving the reproductive performance of late-stage ICR female rats 1. Method 1.1 Laboratory Animals and Grouping Processing 1.1.1 Effects of Traditional Chinese Medicine Formula on Pregnancy Rate and Litter Size in Late-Rating ICR Female Rats Ninety-six late-reproductive ICR female mice were randomly divided into four groups of 24 mice each: a blank control group (Con), a one-week treatment group (TCSF-1W), a two-week treatment group (TCSF-2W), and a three-week treatment group (TCSF-3W). The Con group received 240 mL of autoclaved water daily, while the TCSF-3W group received 12 mL of a traditional Chinese medicine decoction and 228 mL of autoclaved water daily starting from day 1 of the experiment. The TCSF-2W group received the same drug solution from day 8 of the experiment, and the TCSF-1W group received the same drug solution from day 15 of the experiment. Freshly prepared Chinese herbal decoction and autoclaved water were changed daily in each group. On day 22 of the experiment, a total of 24 female ICR mice from each group were divided into 12 cages at a density of 2 mice per cage. According to the principle of female to male ratio of 2:1, 48 male mice were randomly assigned to each cage for mating. After 3 days of mating, the female mice from each group were put together for further observation of pregnancy rate and litter size. 1.1.2 Effects of Traditional Chinese Medicine Formula on Reproductive Function of Late-Rating ICR Female Rats Eighty late-reproductive ICR female mice were randomly divided into four groups of 20 mice each: Con group, TCSF-1W group, TCSF-2W group, and TCSF-3W group. The Con group received 200 mL of autoclaved water daily. The TCSF-3W group received a mixture of 10 mL of herbal decoction and 190 mL of autoclaved water daily from day 1 of the experiment. The TCSF-2W group received the same mixture from day 8, and the TCSF-1W group received the same mixture from day 15. Freshly prepared herbal decoction and autoclaved water were provided daily for each group. On day 22, each mouse was injected with 0.1 mL of 50 IU / mL pregnant horse serum gonadotropin (PMSG) and 0.1 mL of 50 IU / mL human chorionic gonadotropin (hCG). mL, to synchronize estrus and superovulation treatment; after intraperitoneal injection of Zoltil® 50 anesthesia, blood and ovaries were collected from each group of female mice; the ovaries of 10 female mice were removed and placed directly in fixative for subsequent HE staining and sectioning observation; the ovaries of the other 10 female mice were separated from the oviduct dilatation under a stereomicroscope, and oocytes were picked for in vitro fertilization; after the samples were collected, all experimental animals were uniformly anesthetized and euthanized by cervical dislocation, and then disposed of in a harmless manner; 1.2 Composition of Traditional Chinese Medicine Compounds and Preparation of Decoction The traditional Chinese medicine preparation for improving reproductive performance provided in this invention consists of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum, and 10 g of Glycyrrhiza uralensis (processed). The traditional Chinese medicine composition of this invention has the effects of strengthening the spleen and replenishing qi, nourishing yin and blood, warming yang and unblocking collaterals, tonifying the liver and kidneys, and regulating qi and blood, which can improve the reproductive function of male and female mammals as a whole. Take 1000 g of the herbal composition according to the specified ratio, add 3 times the volume of distilled water and soak for 1.5 h; after boiling, simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve; add 3 times the volume of distilled water to the dregs, continue to simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve. Combine the two decoctions and concentrate them over low heat to 1000 mL, filter and prepare a 1 g / mL decoction; administer by drinking water, with a dosage of 0.02 g / kg; 1.3. Statistics on pregnancy rate and litter size in late-reproductive-age ICR female mice The number of pregnant female mice and the number of offspring in each group were recorded, the pregnancy rate was calculated, and the average number of offspring in each group was statistically analyzed to evaluate the effect of the traditional Chinese medicine formula on the reproductive performance of late-stage ICR female mice. 1.4 Statistical observation of fertilized eggs in late-reproductive-age ICR female mice After drug administration, late-reproductive ICR female mice underwent superovulation treatment, and ovarian and oviduct tissues were collected. The ovaries were washed in 37°C pre-warmed PBS to remove adipose tissue, and then transferred to 37°C pre-warmed M2 processing solution. The ampulla of the oviduct was cut open under a stereomicroscope to release the cumulus-oocyte complex. At the same time, epididymal sperm from male mice was collected, capacitated at 37°C and 5% CO2 for 10-15 min, and then co-incubated with the cumulus-oocyte complex in fertilization fluid droplets for 6 h. After fertilization, the cumulus cells were removed with M2 processing solution containing hyaluronidase, and the mice were washed 2-3 times with fresh M2 solution. Finally, the number of fertilized eggs was observed and counted under an inverted microscope. 1.5. Observation of stained sections of ovarian tissue and statistics of follicles at each stage in late-reproductive-age ICR female mice Ovarian tissue was collected from late-reproductive-age ICR female mice in each group. After fixation, the tissue was trimmed into approximately 5 × 5 × 2 mm blocks and rinsed overnight in running water. Subsequently, the tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then cut into 4 μm thick paraffin sections. After dewaxing and rehydration with graded ethanol, the sections were stained with hematoxylin and eosin and mounted with neutral resin. After staining, the morphology of the ovarian tissue was observed under an upright microscope, and the number of primordial follicles, primary follicles, secondary follicles, mature follicles, and corpora lutea was counted to evaluate the effect of the traditional Chinese medicine formula on follicular development in late-reproductive-age ICR female mice. 1.6 Detection of peripheral blood reproductive-related hormone levels in late-reproductive-age ICR female mice Peripheral blood was collected from late-reproductive ICR female mice in each group. After centrifugation at 3000 r / min, the supernatant serum was collected. Following the ELISA kit instructions, the standard or serum sample was added to a pre-coated 96-well plate, followed by the addition of HRP-labeled detection antibody. The plate was incubated at 37°C for 60 min. After washing, substrate solution was added for color development, and the plate was incubated at 30°C in the dark for 15 min. The reaction was then terminated by adding stop solution, and the OD value was read at 450 nm. The levels of follicle-stimulating hormone (FSH) and estradiol (E2) in the samples were calculated based on the standard curve. 1.7 Detection of peripheral blood oxidative stress-related indicators in late-reproductive-age ICR female rats Peripheral blood was collected from late-reproductive-age ICR female mice, and serum was obtained by centrifugation. Oxidative stress-related indicators were detected according to the corresponding kit instructions. Serum samples were used to determine superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, and malondialdehyde (MDA) content. SOD was measured at 450 nm after incubation at 37℃; GSH-PX was measured at 412 nm after reaction at room temperature; and MDA was measured at 532 nm after reaction in a 95℃ water bath, cooling, and centrifugation. The levels of each indicator were calculated according to the kit instructions to evaluate the effect of the traditional Chinese medicine formula on the oxidative stress status of late-reproductive-age ICR female mice. 1.8 Data Analysis Statistical analysis was performed using IBM SPSS Statistics 17.0 software, and graphs were created using Graphpad Prism 9.5.1. All data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons between groups. The criterion for statistical significance was... P <0.05.

[0034] 2. Experimental Results 2.1 Pregnancy rate and litter size in late-reproductive-age ICR female mice in each experimental group like Figure 11 As shown, compared with the Con group, the pregnancy rate of female mice in the TCSF-2W group was significantly higher. P < 0.05); the pregnancy rate of female mice in the TCSF-3W group was significantly increased ( P< 0.01); there was no significant difference in litter size between female mice in the TCSF-1W and TCSF-2W groups, while the litter size in the TCSF-3W group was significantly increased. P < 0.01); 2.2 Number of fertilized eggs after in vitro fertilization in late-reproductive-age ICR female mice in each experimental group like Figure 12 As shown, the number of fertilized eggs in female mice in the Con group was 6, the number of fertilized eggs in female mice in the TCSF-1W group was 8, the number of fertilized eggs in female mice in the TCSF-2W group was 10, and the number of fertilized eggs in female mice in the TCSF-3W group was 12; the number of fertilized eggs in female mice gradually increased with the duration of drug administration. 2.3 Observation of stained sections of ovarian tissue and statistics of follicles at each stage in late-reproductive-age ICR female mice of different experimental groups like Figure 13 As shown in Table 2, compared with the CON group, the number of corpora lutea on the sections of female mice in the TCSF-1W group was not significantly different, while the number of corpora lutea on the sections of female mice in the TCSF-2W group was significantly increased. P < 0.01), the number of corpora lutea on sections of female mice in the TCSF-3W group was significantly increased ( P < 0.001). There were no significant differences in the number of primordial follicles, primary follicles, secondary follicles, mature follicles, and atretic follicles on the sections of female mice in the TCSF-1W, TCSF-2W, and TCSF-3W groups. Table 2. Number of different cell types on ovarian sections of late-reproductive-age ICR male rats in each group. ; Note: Data are the mean ± standard deviation of 4 parallel samples; compared with the Con group, *** P < 0.001, ** P <0.01;

[0035] 2.4 Peripheral blood levels of reproductive-related hormones in late-reproductive-age ICR female rats in different experimental groups like Figure 14 As shown, compared with the Con group mice, the E2 level of female mice in the TCSF-1W group was not significantly different; the E2 level of female mice in the TCSF-2W group was significantly increased ( P < 0.05); E2 levels in female mice in the TCSF-2W group were significantly increased ( P < 0.01); Compared with the Con group mice, the FSH levels of female mice in the TCSF-1W group were not significantly different, while the FSH levels of female mice in the TCSF-2W and TCSF-3W groups were significantly decreased ( P < 0.05); 2.5 Peripheral blood oxidative stress-related indicators in late-reproductive-age ICR female rats in different experimental groups like Figure 15 As shown, compared with the Con group mice, TCSF treatment reduced oxidative damage and significantly improved antioxidant capacity; the MDA content of female mice in the TCSF-1W group was significantly reduced ( P < 0.05), SOD and GSH-Px activities were significantly increased ( P <0.05); the activities of SOD and GSH-Px in female mice in the TCSF-2W group were significantly increased ( P < 0.05), MDA content decreased significantly ( P <0.05); the activities of SOD and GSH-Px in female mice in the TCSF-3W group were significantly increased ( P < 0.01), MDA content decreased significantly ( P <0.01); 3. Summary The traditional Chinese medicine formula significantly improved the pregnancy rate, litter size, and number of in vitro fertilized eggs in late-reproductive-age ICR female mice, promoted follicle development and corpus luteum formation, and improved peripheral blood reproductive hormone levels and oxidative stress. Its effects increased with prolonged administration time, suggesting that the formula can improve the reproductive performance of late-reproductive-age ICR female mice by regulating reproductive endocrine function, enhancing antioxidant capacity, and improving ovarian function.

[0036] Example 4: Effects of different traditional Chinese medicine compositions on improving reproductive performance in late-breeding ICR male rats 1. Method 1.1 Laboratory Animals and Grouping Processing Forty late-stage ICR male rats were randomly divided into four groups of ten each: Con group, TCSF-1W group, TCSF-2W group, and TCSF-3W group. The Con group received 100 mL of autoclaved water daily. The TCSF-3W group received a mixture of 5 mL of herbal decoction and 95 mL of autoclaved water daily from day 1 of the experiment. The TCSF-2W group received the same mixture from day 8 of the experiment, and the TCSF-1W group received the same mixture from day 15 of the experiment. Freshly prepared herbal decoction and autoclaved water were provided daily for each group. On day 22 of the experiment, blood and epididymis were collected from the male rats after intraperitoneal injection of Zoltil® 50. The epididymis of five male rats were used for sperm motility testing, and the epididymis of the other five male rats were immediately placed in fixative for subsequent HE staining and sectioning. After the samples were collected, all experimental animals were euthanized by cervical dislocation under anesthesia and then disposed of in a harmless manner. 1.2 Composition of Traditional Chinese Medicine Compounds and Preparation of Decoction The traditional Chinese medicine preparation for improving reproductive performance provided in this invention consists of 15 g of Codonopsis pilosula, 15 g of Astragalus membranaceus, 10 g of Atractylodes macrocephala, 20 g of Poria cocos, 10 g of Magnolia officinalis, 10 g of Ophiopogon japonicus, 6 g of Cinnamomum cassia, 6 g of Paeonia lactiflora, 3 g of Cinnamomum cassia, 20 g of Lycium barbarum, and 10 g of Glycyrrhiza uralensis (processed). The traditional Chinese medicine composition of this invention has the effects of strengthening the spleen and replenishing qi, nourishing yin and blood, warming yang and unblocking collaterals, tonifying the liver and kidneys, and regulating qi and blood, which can improve the reproductive function of male and female mammals as a whole. Take 1000 g of the herbal composition according to the specified ratio, add 3 times the volume of distilled water and soak for 1.5 h; after boiling, simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve; add 3 times the volume of distilled water to the dregs, continue to simmer over low heat for 2 h, and filter the decoction through an 80-mesh sieve. Combine the two decoctions and concentrate them over low heat to 1000 mL, filter and prepare a 1 g / mL decoction; administer by drinking water, with a dosage of 0.02 g / kg; 1.3. Sperm motility detection in late-reproductive-age ICR male rats The collected epididymis was placed in PBS preheated to 37°C. The epididymis was carefully removed, and a cross-shaped incision was made in it with scissors. The epididymis was then immersed in capacitation solution preheated to 37°C to allow sperm to swim out naturally. After 3 minutes, 5 μL of semen sample was collected and added to a Markler sperm counting chamber. Three clear fields of view were randomly selected. Sperm motility and various movement data were analyzed using software. Incorrectly labeled sperm were manually corrected, and the results were recorded. The parameters detected included curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), total motor sperm rate (TM), linearity index (LIN), and straightness index (STRAIL). STR) and vibration index (Wobble, WOB); 1.4 Observation of stained sections of epididymal tissue from late-reproductive-age ICR male rats Epididymal tissue was collected from late-stage ICR male rats in each group. After fixation, the tissue was trimmed into approximately 5 × 5 × 2 mm blocks and rinsed overnight in running water. Subsequently, the tissue was dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, and then cut into 4 μm thick paraffin sections. After dewaxing and rehydration with graded ethanol, the sections were stained with hematoxylin-eosin and mounted with neutral resin. After staining, the structure of the vas deferens and sperm density in the epididymis of male rats were observed under an upright microscope to evaluate the effect of the traditional Chinese medicine formula on the epididymal development of young ICR male rats. 1.5 Detection of peripheral blood reproductive-related hormone levels in late-reproductive-age ICR male rats Peripheral blood was collected from young male ICR rats in each group. After centrifugation at 3000 r / min, the supernatant serum was collected. Following the ELISA kit instructions, standards or serum samples were added to pre-coated 96-well plates, followed by the addition of HRP-labeled detection antibody. The plates were incubated at 37°C for 60 min. After washing, substrate solution was added for color development, and the plates were incubated at 30°C in the dark for 15 min. The reaction was then terminated with stop solution, and the OD value was read at 450 nm. The testosterone (T) level in the peripheral blood of late-reproductive-age ICR male rats was calculated based on the standard curve. 1.6 Detection of peripheral blood oxidative stress-related indicators in late-reproductive-age ICR male rats Peripheral blood was collected from late-reproductive-age ICR male rats and serum was obtained by centrifugation. Oxidative stress-related indicators were detected according to the corresponding kit instructions. Serum samples were used to determine superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-PX) activity, and malondialdehyde (MDA) content. SOD was detected by incubation at 37℃ and OD value was read at 450 nm. GSH-PX was detected by reaction at room temperature and OD value was read at 412 nm. MDA was detected by reaction in a 95℃ water bath, cooling and centrifugation and OD value was read at 532 nm. The levels of each indicator in peripheral blood of late-reproductive-age ICR male rats were calculated according to the kit instructions to evaluate the effect of the traditional Chinese medicine formula on the oxidative stress status of young ICR male rats. 1.7 Data Analysis Statistical analysis was performed using IBM SPSS software, and graphs were created using Graphpad Prism 9.5.1. Data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons between groups. The criterion for statistical significance was... P <0.05.

[0037] 2. Experimental Results 2.1 Sperm quality in different experimental groups of late-reproductive-age ICR male rats like Figure 16 As shown, compared with the Con group, the overall sperm motility of male mice treated with TCSF showed an improving trend. There were no significant differences in ALH, VAP, VSL, and STR levels in the sperm of male mice in the TCSF-1W, TCSF-2W, and TCSF-3W groups. There were no significant differences in VCL levels in the sperm of male mice in the TCSF-1W and TCSF-2W groups, but the TCSF-3W group showed a significant increase. P < 0.05); there was no significant difference in sperm BCF in male mice in the TCSF-1W group, while it was significantly increased in the TCSF-2W and TCSF-3W groups ( P < 0.01); There was no significant difference in TM in male mice in the TCSF-1W group, but it was significantly increased in the TCSF-2W group ( P < 0.05), the TCSF-3W group showed a highly significant improvement ( P < 0.01); there was no significant difference in sperm LIN in male mice in the TCSF-1W group, but it was significantly reduced in the TCSF-2W group ( P < 0.05), the TCSF-3W group showed a highly significant decrease ( P < 0.01) There was no significant difference in WOB (whole body volume) of sperm in male mice in the TCSF-1W group, while it was significantly reduced in both the TCSF-2W and TCSF-3W groups. P < 0.05); like Figure 17 As shown, compared with the Con group, the sperm density and sperm motility of male mice were improved to varying degrees after TCSF treatment. With the increase of the feeding time of Chinese medicine, the number of dead sperm decreased, the proportion of low-speed sperm decreased, and the proportion of medium-speed and high-speed sperm increased. 2.2 Observation of epididymal tissue sections stained from different experimental groups of late-fertile ICR male rats like Figure 18 As shown, compared with the CON group of male rats, the vas deferens lumen filling degree of the epididymis was significantly increased after TCSF treatment, and the proportion of sperm in the lumen was significantly increased. 2.3 Peripheral blood levels of reproductive-related hormones in late-reproductive-age ICR male rats in different experimental groups like Figure 19 As shown, compared with the Con group mice, there was no significant difference in T content in male mice in the TCSF-1W group; however, the T content in male mice in the TCSF-2W group was significantly increased. P < 0.05); T content was significantly increased in male mice in the TCSF-3W group ( P < 0.01); 2.4 Peripheral blood oxidative stress-related indicators in late-reproductive-age ICR male rats in different experimental groups like Figure 20As shown, compared with the Con group mice, TCSF treatment reduced oxidative damage and significantly improved antioxidant capacity; the MDA content in male mice in the TCSF-1W group was significantly reduced ( P < 0.05), GSH-Px activity was significantly increased ( P <0.05), there was no significant difference in SOD activity; in the TCSF-2W group, the SOD and GSH-Px activities of male mice were significantly increased ( P < 0.05), MDA content was significantly decreased (P < 0.05); SOD and GSH-Px activities were significantly increased in male mice in the TCSF-3W group (P < 0.05). P < 0.01), MDA content decreased significantly ( P < 0.01).

[0038] 3. Summary The traditional Chinese medicine formula can improve sperm motility and sperm density in late-stage ICR male rats, improve epididymal tissue morphology, increase testosterone levels, and enhance antioxidant capacity and reduce oxidative damage; its improvement effect increases with the duration of administration.

Claims

1. A traditional Chinese medicine composition for improving reproductive performance, characterized in that, The traditional Chinese medicine composition comprises the following components in parts by weight: Codonopsis pilosula 14-16 parts, Astragalus membranaceus 14-16 parts, Atractylodes macrocephala 8-12 parts, Poria cocos 18-22 parts, Magnolia officinalis 8-12 parts, Ophiopogon japonicus 8-12 parts, Cinnamomum cassia 5-7 parts, Paeonia lactiflora 5-7 parts, Cinnamomum cassia 2-4 parts, Lycium barbarum 18-22 parts, and Glycyrrhiza uralensis (processed) 8-12 parts.

2. The method for preparing the traditional Chinese medicine composition for improving reproductive performance according to claim 1, characterized in that, The preparation steps are as follows: Step (1) Soak the raw materials to be decocted in 2 to 4 times their volume of distilled water according to the proportion; wherein the soaking time is 1 to 2 hours. Step (2) After soaking, bring the soaked raw materials to a boil over high heat. After boiling, reduce to a simmer and continue simmering. After simmering, filter the dregs through an 80-mesh sieve and collect the liquid. The filtered dregs will be simmered again. The simmering time is 1.5 to 2.5 hours. Step (3) Add 2 to 4 times the volume of distilled water to the filtered residue, bring to a boil over high heat, then reduce to a simmer and continue simmering. After simmering, filter the residue through an 80-mesh sieve and discard it, then collect the liquid. Combine the two decoctions and simmer over low heat to concentrate the liquid to the same volume as the raw materials to make the final decoction. The simmering time is 1.5 to 2.5 hours.

3. The application of the traditional Chinese medicine composition according to claim 1 in the preparation of a product that improves reproductive performance.

4. The application according to claim 3, characterized in that, The applications include the use in the preparation of drugs to improve pregnancy rates and litter sizes in young and late-reproductive-age ICR female mice.

5. The application according to claim 3, characterized in that, The applications include the use in the preparation of drugs to improve in vitro fertilization after overovulation in young and late-reproductive-age ICR female mice.

6. The application according to claim 3, characterized in that, The applications include the use in the preparation of drugs to improve follicle development in young and late-reproductive-age ICR female mice.

7. The application according to claim 3, characterized in that, The applications include the use in the preparation of drugs to improve sperm quality in young and late-reproductive-age ICR male rats.

8. The application according to claim 3, characterized in that, The applications include the use in the preparation of drugs that improve the morphology of epididymal tissue in young and late-fertile ICR male rats.

9. The use of the traditional Chinese medicine composition according to claim 3 in the preparation of a drug for improving the levels of reproductive-related hormones in the peripheral blood of young and late-reproductive-age ICR female and male mice.

10. The application of the traditional Chinese medicine composition according to claim 3 in the preparation of a drug for improving peripheral blood oxidative stress indicators in young and late-reproductive-age ICR female and male mice.

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