A cervus nippon synchronously estrous male deer intraspecific mating species twin regulation agent and application method

Abstract

This invention relates to the field of animal reproductive regulation technology, and discloses a twinning regulator for sika deer males that synchronize estrus and its application method. The regulator is a homogeneous and transparent sol system prepared from trehalose, pituitary follicle-stimulating hormone (FSH), medium- and low molecular weight sodium hyaluronate, low molecular weight dextran sulfate, zinc gluconate, and physiological saline. This invention utilizes sodium hyaluronate of a specific molecular weight and dextran sulfate to construct a polysaccharide sol network, combined with the cross-linking effect of zinc gluconate and the physicochemical stabilizing effect of trehalose, to encapsulate and slowly release FSH. The application method includes implanting and removing a vaginal silicone plug, combined with the injection of pregnant mare serum gonadotropin (PMSG) and the regulator, followed by natural mating. This invention prolongs the half-life of exogenous hormones in the animal, avoids drastic fluctuations in blood drug concentration, and improves the stability of twinning rates in sika deer.

Description

Technical Field

[0001] This invention relates to the field of animal reproductive regulation technology, specifically to a twin pregnancy regulator for sika deer males that synchronize estrus and its application method. Background Technology

[0002] In the sika deer farming industry, artificial intervention to improve the reproductive rate and twinning rate of does is a way to increase economic benefits. Currently, the common practice in farming is to inject exogenous reproductive hormones combined with estrus synchronization techniques to promote follicle development in does. Existing exogenous hormone preparations are mostly conventional aqueous solutions. After injection into the does's muscle tissue, the drug diffuses and is metabolized rapidly, causing fluctuations in blood drug concentration within a short period. These fluctuations in blood drug concentration make it difficult to control the number of follicles developing. An excessively high initial concentration peak can stimulate the ovaries, leading to multiple ovulations. Subsequently, the concentration drops rapidly, failing to maintain the continuous maturation of two dominant follicles, resulting in an unstable twinning rate in sika deer.

[0003] Meanwhile, protein hormones such as pituitary follicle-stimulating hormone (FSH) exhibit low physicochemical stability in traditional liquid formulations. During storage and after injection into animal tissues, protein molecules undergo conformational unfolding and degradation due to thermodynamic changes or the influence of in vivo enzymes. This results in a short half-life for exogenous protein hormones in animals, low overall bioavailability of the formulation, and an inability to provide stable drug concentration support for follicle development.

[0004] Regarding the integration of drug administration procedures with mating protocols, existing estrus synchronization intervention techniques lack design tailored to the kinetics of reproductive hormone uptake. Conventional drug administration fails to combine long-acting and short-acting hormones or administer them at specific sites, making it difficult to regulate the concentrated ovulation window of the dominant follicles in the female deer over time. This results in a mismatch between the actual ovulation time and the sperm capacitation time in the reproductive tract during natural mating in the male deer, reducing the probability of fertilization and hindering the overall conception rate of the herd. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a twinning regulator for sika deer males that synchronize estrus and their application method. This invention solves the problems of unstable twinning rates and overall conception rates in sika deer caused by the rapid diffusion of conventional aqueous reproductive hormone preparations after injection, leading to fluctuations in blood drug concentration and resulting in multiple ovulations; the easy degradation of protein hormones and their short half-life in vivo; and the difficulty in matching the existing dosing procedures with the ovulation window and natural mating time.

[0006] To achieve the above objectives, the present invention provides a twinning regulator for sika deer males that synchronize estrus, employing the following technical solution: A twin pregnancy regulator for sika deer males that are in estrus synchronously, wherein the twin pregnancy regulator is a homogeneous and transparent sol system; The twin pregnancy regulator is made from the following raw material components: 200-500 mg trehalose, 50-200 IU pituitary follicle-stimulating hormone, 100-400 mg medium and low molecular weight sodium hyaluronate, 20-100 mg low molecular weight dextran sulfate and 1.8-14 mg zinc gluconate per 8-12 mL system. Furthermore, the twin pregnancy regulator is diluted to volume with physiological saline at a mass concentration of 0.8%-1.0%, and the pH value of the twin pregnancy regulator is 7.2-7.4; The sodium hyaluronate of medium and low molecular weight has a weight-average molecular weight of 10kDa-50kDa, the dextran sulfate of low molecular weight has a weight-average molecular weight of 36kDa-50kDa, and the zinc gluconate provides a final zinc ion concentration of 0.5-2.5 mmol / L in the system.

[0007] By employing the above technical solution, a sol matrix was constructed using sodium hyaluronate of a specific molecular weight range and low molecular weight dextran sulfate, supplemented with zinc gluconate and trehalose, to establish a microenvironment for the encapsulation and sustained release of pituitary follicle-stimulating hormone. This resulted in extending the hormone's in vivo half-life, improving bioavailability, and achieving a stable twin pregnancy rate. The specific mechanism of action is as follows: Step 1: The zinc ions generated by the system dissolution have suitable charge density and coordination ability. Zinc ions interact with histidine residues and free carboxyl groups on the surface of pituitary follicle-stimulating hormone (FSH) through polydentate coordination, forming a structurally stable protein-metal ion complex. This reduces the conformational flexibility of FSH in the aqueous phase and restricts its spatial unfolding caused by thermal or enzymatic degradation.

[0008] Step Two: Low molecular weight dextran sulfate with a weight-average molecular weight of 36kDa-50kDa carries a high density of negatively charged sulfate groups, while medium-low molecular weight sodium hyaluronate with a weight-average molecular weight of 10kDa-50kDa carries carboxyl groups. Both fully extend in a physiological saline environment with a pH of 7.2-7.4. Zinc ions act as bridging hubs, binding to protein molecules at one end and electrostatically cross-linking and coordinating with the carboxyl groups of sodium hyaluronate and the sulfate groups of dextran sulfate at the other end. This process promotes the entanglement of linear polysaccharide molecular chains, forming a thixotropic homogeneous transparent sol three-dimensional network. Pituitary follicle-stimulating hormone molecules are physically trapped and anchored within the micropores of this sol network.

[0009] Step 3: Trehalose molecules contain a large number of non-reducing hydroxyl groups, which replace water molecules in a hydrogen bond network to form a dense hydration shell on the surface of pituitary follicle-stimulating hormone (FSH). Simultaneously, trehalose molecules embed themselves in the polysaccharide sol network, increasing the local viscosity of the sol matrix and reducing the freedom of movement of free water molecules. When the regulator is injected into the muscle tissue of the doe, tissue fluid seeps into the sol network, causing the polysaccharide matrix to degrade and swell. The encapsulated FSH-zinc complex diffuses into the bloodstream at a constant rate. This maintains a stable blood drug concentration, avoiding excessive stimulation of the ovaries caused by fluctuations in peak blood drug levels.

[0010] Preferably, in the raw material components of the twin pregnancy regulator, the mass ratio of the medium-low molecular weight sodium hyaluronate to the low molecular weight dextran sulfate is 3.0-4.5:1, and the mass ratio of the trehalose to the medium-low molecular weight sodium hyaluronate is 1.0-1.5:1.

[0011] By employing the above technical solution, controlling the mass ratio of sodium hyaluronate to dextran sulfate at 3.0-4.5:1 allows for the establishment of an electrostatic equilibrium between carboxyl and sulfate groups in the sol-gel network. If the proportion of dextran sulfate is too high, excess anions can cause a shift in the isoelectric point of follicle-stimulating hormone, resulting in localized sedimentation. If the proportion of sodium hyaluronate is too high, the pore size of the polysaccharide network increases, leading to a decrease in hormone retention capacity and a burst release phenomenon. Limiting the ratio of trehalose to sodium hyaluronate to 1.0-1.5:1 allows small-molecule sugars to form hydrogen-bonded filling layers between the polysaccharide polymer backbone, endowing the system with suitable microrheological properties, facilitating syringe injection, and rapidly forming an in-situ retained gel reservoir in the interstitial space after injection.

[0012] Preferably, the medium-to-low molecular weight sodium hyaluronate is prepared by the following steps: Take high molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000kDa-1500kDa, dissolve it in phosphate buffer solution with a pH of 6.0-6.5, and prepare a solution with a mass fraction of 0.8%-1.2%. Add hyaluronidase at a dosage of 1000-5000 U / g of substrate to the solution, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 35-40℃ for 2-6 hours to obtain the reaction solution; The reaction solution was heated to 85-95℃ and held for 8-15 minutes to inactivate the enzyme, resulting in an inactivated reaction solution. The inactivated reaction solution was centrifuged to collect the supernatant, which was then purified in two stages using a tangential flow ultrafiltration system. First, the primary filtrate was collected through an ultrafiltration membrane with a molecular weight cutoff of 50-100 kDa. Then, the primary filtrate was passed through an ultrafiltration membrane with a molecular weight cutoff of 5-30 kDa to collect the retentate. The retentate was dialyzed to remove salts and then freeze-dried to obtain the medium-low molecular weight sodium hyaluronate.

[0013] By employing the above-mentioned technical solution, hyaluronidase is used for specific endoglycosidic bond hydrolysis, which can cleave the β-1,4-glycosidic bonds in the backbone of macromolecular polysaccharides. Compared with chemical degradation under strong acid-base systems, enzymatic degradation avoids destroying the monosaccharide pyran ring structure and characteristic carboxyl functional groups inside the hyaluronic acid molecule. Combined with a two-stage tangential flow ultrafiltration separation process, products in the target molecular weight range of 10kDa-50kDa are precisely retained. This eliminates the risk of immune inflammation in animal tissues caused by extremely low molecular weight oligosaccharide fragments and reduces the fluid viscosity resistance caused by high molecular weight components, providing standardized functional units for the subsequent construction of a structurally homogeneous sol matrix.

[0014] Preferably, the low molecular weight sulfated dextran is prepared by the following steps: Dextran T70 was placed in a 0.08-0.12 mol / L hydrochloric acid solution and subjected to acid hydrolysis at 55-65℃ for 1-3 hours. When the weight-average molecular weight decreased to 18-25 kDa, the reaction was terminated by neutralizing with a 0.8-1.2 mol / L sodium hydroxide solution to a pH of 6.8-7.2, yielding the reaction termination solution. The reaction termination solution was precipitated with anhydrous ethanol and vacuum dried to obtain a low molecular weight dextran intermediate; The low molecular weight dextran intermediate was dissolved in N,N-dimethylformamide to form a solution, and then anhydrous pyridine was added to the solution at a volume ratio of 0.8-1.2:1. The solution was then modified by sulfation by dropwise addition of a chlorosulfonic acid-pyridine complex at 55-65°C for 2-4 hours to obtain a modified solution. The mass ratio of chlorosulfonic acid to the low molecular weight dextran intermediate was 1.8-2.2:1. The modified solution was dialyzed with deionized water for 40-50 hours, and then freeze-dried to obtain the low molecular weight dextran sulfate.

[0015] By employing the above-mentioned technical solution, hydrochloric acid is used to catalyze the cleavage of α-1,6-glycosidic bonds in the polysaccharide backbone, reducing the molecular weight from the 70 kDa range to a predetermined range of 18-25 kDa. In the subsequent sulfation modification system, chlorosulfonic acid undergoes an electrophilic substitution reaction under the proton-accepting catalysis of anhydrous pyridine. The free hydroxyl groups at the glucose residue sites in the dextran molecule undergo esterification with sulfate groups, generating a polysaccharide sulfate ester structure and releasing hydrogen chloride as a byproduct. These reaction parameters lock the final product's weight-average molecular weight and degree of sulfation within the range of 36 kDa-50 kDa, endowing the polysaccharide molecular chain with a high density of negative charge, enabling it to electrostatically bind to cationic regions on protein surfaces.

[0016] Preferably, the twin pregnancy regulator is prepared by the following steps: Measure 7-9 mL of 0.8%-1.0% physiological saline into a sterile mixing tank, add trehalose, and dissolve it by magnetic stirring to obtain the initial trehalose solution. Add sodium dihydrogen phosphate-disodium hydrogen phosphate buffer dropwise to the initial trehalose solution to adjust the pH of the system to 7.2-7.4 to obtain the base solution. Turn on the cooling circulation system to cool the base solution and maintain it at 2℃-8℃. Under constant temperature conditions of 2℃-8℃, the lyophilized pituitary follicle-stimulating hormone powder was added to the base solution and stirred at 50-150 rpm to dissolve it, thus obtaining a preliminary solution system. While maintaining a constant temperature, the low molecular weight dextran sulfate and the medium-low molecular weight sodium hyaluronate are sequentially dispersed into the initial dissolution system, and the mixture is continuously stirred at a low speed until the powder dissolves to form a sol system. The sol system was vortexed at 1000-2500 rpm for 5-15 minutes, during which a pre-prepared zinc gluconate aqueous solution was added dropwise to achieve a zinc ion concentration of 0.5-2.5 mmol / L, thus obtaining a zinc-containing sol. The zinc-containing sol was then brought to a final volume of 8-12 mL with 0.8%-1.0% physiological saline, and the mixture was continuously vortexed to obtain a homogeneous regulator sol. The homogeneous regulator sol was dispensed into sterile syringes, sealed, and stored at 2℃-8℃ protected from light for later use.

[0017] By employing the above technical solution, the entire preparation process is conducted in a low-temperature environment of 2℃-8℃ and a buffer environment with a pH of 7.2-7.4, which inhibits the thermodynamic unfolding and isoelectric point aggregation and dehydration of pituitary follicle-stimulating hormone molecules. Through controlled feeding sequence, trehalose is pre-dissolved to establish a highly permeable hydration medium, followed by the dissolution of protein molecules, and finally the addition of polyelectrolyte polysaccharides. This avoids the direct adsorption of water by the dry powdered polysaccharides, which could lead to localized protein dehydration and denaturation. A high-frequency vortex flow field accompanied by the slow dripping of zinc ions promotes the uniform penetration of metal ions into the polysaccharide network pores, resulting in in-situ cross-linking reactions and preventing polysaccharide flocculation caused by localized zinc ion overload. Fluid dynamic control, transitioning from low-speed stirring to high-frequency oscillation, promotes the orderly extension of long polysaccharide chains under mild shear stress, ultimately yielding a phase-stable, homogeneous sol-gel reservoir system free of visible particles.

[0018] Secondly, this invention provides a method for applying a twin pregnancy regulator for sika deer males that synchronize estrus, employing the following technical solution: A method for applying a twin-birth regulator for sika deer estrus-synchronizing males in the first aspect, comprising the following steps: A vaginal silicone plug containing progesterone was implanted deep into the vagina of a healthy female sika deer, and the day of implantation was recorded as day 0. The vaginal silicone plug was removed 12-14 days after implantation. At the same time as plug removal, pregnant mare serum gonadotropin and the twin pregnancy regulator were injected into the muscle area of ​​the mother sika deer. 36-44 hours after the thrombectomy and drug administration, the female sika deer that has been injected with the twin pregnancy regulator is placed in the breeding pen, and a healthy male deer is introduced for natural mating.

[0019] By employing the above-mentioned technical approach, and using a synergistic regulatory method combining progesterone suppository treatment with dual administration of exogenous hormones, an intervention program targeting the follicular development and ovulation cycle of female sika deer was established. This program achieved the effects of shortening the estrus-mating cycle, increasing the double ovulation rate, and improving the conception rate. The specific mechanism of action is as follows: Step 1: During the implantation period, the progesterone in the vaginal silicone plug is continuously absorbed into the bloodstream through the vaginal mucosa, increasing the progesterone concentration in the does's body fluids. This exerts a negative feedback inhibitory effect on the hypothalamus and pituitary gland, blocking the secretion of endogenous gonadotropins. This process causes follicles at different physiological stages in the does to cease development, achieving basic synchronization of the estrous cycles across the entire herd.

[0020] Step Two: On days 12-14, the vaginal silicone plug is removed. This reduces progesterone levels in the does, relieving negative feedback inhibition. Simultaneously, pregnant mare serum gonadotropin (PMS) and the aforementioned twin pregnancy regulator are administered intramuscularly. PMS has long-acting follicle-stimulating and luteinizing dual biological activities, entering the bloodstream to initiate the recruitment and development of follicle clusters. The sol system in the twin pregnancy regulator forms an in-situ reservoir in the interstitial space, where pituitary follicle-stimulating hormone is slowly released into the bloodstream, creating a long-acting and short-acting pharmacokinetic complementarity with PMS. A constant, low concentration of exogenous follicle-stimulating hormone replenishes the threshold hormone levels required for follicle development, promoting synchronous maturation of the two dominant follicles and avoiding the excessively high hormone spikes caused by a single injection of the liquid formulation, which could lead to multiple ovulation.

[0021] Step 3: 36-44 hours after thrombectomy and drug administration, the two dominant follicles in the ovary complete development and enter the concentrated ovulation window. At this time, a male deer is introduced for natural mating. The time when the sperm capacitation in the reproductive tract coincides with the time when the two eggs are released, increasing the twin fertilization rate.

[0022] Preferably, before implanting the vaginal silicone plug, the vulva of the female sika deer is disinfected and pretreated with a 0.08%-0.15% potassium permanganate solution.

[0023] By employing the above-mentioned technical solution, potassium permanganate dissociates into permanganate ions in aqueous solution. These ions, with their strong oxidizing properties, disrupt the cell membrane structure and enzyme system of pathogenic microorganisms attached to the vulva, reducing the density of contact pathogens. This pretreatment interrupts the delivery pathway of external environmental bacteria carried into the deep vagina by the suppository applicator during the silicone suppository insertion procedure, reducing the risk of inflammatory reactions in the reproductive tract mucosa.

[0024] Preferably, the pregnant mare serum gonadotropin is injected into the neck muscle of the female sika deer at a dose of 330-350 IU.

[0025] By employing the above-mentioned technical solution, the neck muscle group has a dense capillary distribution and high blood flow. After pregnant mare serum gonadotropins are injected into this area, they can be transported across the membrane along the concentration gradient into the systemic circulation. The dosage threshold of 330-350 IU meets the initial hormone receptor occupancy requirements for the initiation of follicular wave development in sika deer, which can both avoid basal follicular atresia and prevent excessively high substrate concentrations from interfering with the controlled release of subsequent twin pregnancy regulators.

[0026] Preferably, the twin pregnancy regulator is injected into the deep muscle of the buttocks of the female sika deer, with an injection dosage of 1.5-2.5 mL / vial.

[0027] By employing the above-mentioned technical solution, the flow rate of tissue fluid in the deep muscles of the buttocks is relatively slow, and the contraction amplitude of muscle fibers is less than that in the neck, providing a highly stable microenvironment for the homogeneous transparent sol system after injection. The limited administration volume of 1.5-2.5 mL ensures that the polysaccharide cross-linked network forms a gel reservoir with a suitable specific surface area in the intermuscular bundles. This keeps the enzymatic swelling rate of glycosidic bonds within the matrix constant, ensuring that pituitary follicle-stimulating hormone diffuses at a uniform rate and maintains an effective blood drug concentration for tens of hours.

[0028] This invention provides a twin pregnancy regulator for sika deer males that synchronize estrus and a method for its application. It has the following beneficial effects: 1. The twin pregnancy regulator of the present invention utilizes a sol matrix constructed from medium and low molecular weight sodium hyaluronate and low molecular weight dextran sulfate, and encapsulates pituitary follicle-stimulating hormone (FSH) through the cross-linking effect of zinc gluconate. This structure alters the metabolic pathway of rapid drug diffusion after injection of conventional aqueous formulations, allowing the hormone to be released slowly at a constant rate in the muscle tissue of the female deer. This avoids drastic fluctuations in blood drug concentration caused by a single injection, thus satisfying the hormone requirements for twin follicle development and preventing multiple ovulation caused by excessively high concentrations, thereby improving the stability of twin pregnancy rate in sika deer.

[0029] 2. This invention improves the physicochemical stability of pituitary follicle-stimulating hormone by introducing trehalose and zinc gluconate into the regulator component. Trehalose replaces water molecules to form a hydration layer on the outside of the hormone protein, while zinc ions restrict changes in its spatial conformation by coordinating with protein surface residues. The combined effect of the polysaccharide matrix and the above-mentioned stabilizers reduces the degradation loss of the protein hormone during low-temperature storage and after injection into animals, prolongs the half-life of exogenous hormones, and improves the overall bioavailability of the formulation.

[0030] 3. The application method of this invention achieves synchronized estrus and precise mating of a herd of does through a combination of vaginal silicone plugs and multiple exogenous hormones. By injecting pregnant mare serum gonadotropin into the neck and a twin pregnancy regulator into the deep muscle of the buttocks when the silicone plug is removed, the difference in absorption rate at different sites creates a complementary effect of long-acting and short-acting hormones, controlling the maturation and concentrated ovulation window of the two dominant follicles, so that it coincides with the time of natural mating of the male deer, thereby increasing the probability of sperm-egg fusion in the reproductive tract and the overall conception rate. Attached Figure Description

[0031] Figure 1 This is a graph showing the change in apparent viscosity of each group of drug solutions with shear rate at 5°C in Test Example 1 of the present invention. Figure 2 This is a comparison graph of the apparent viscosity of each group of drug solutions under 5°C conditions in Test Example 1 of the present invention; Figure 3 This is a graph showing the change in the cumulative exudation rate of trehalose in simulated tissue fluid at 38.5℃ over time in Example 2 of Test Example 2 of the present invention. Figure 4 This is a bar chart showing the peak thrust of the formulation in Test Example 3 of the present invention at a constant injection rate of 5°C. Figure 5 This is a bar chart showing the peak thrust of the formulation in the comparative example of Test Example 3 of the present invention at a constant injection rate of 5°C. Figure 6 This is a graph showing the cumulative release rate of pituitary follicle-stimulating hormone in each group of formulations in Test Example 4 of the present invention in an in vitro release model at 38.5°C. Figure 7 This is a bar chart showing the estrus synchronization rate and twinning rate of each group of sika deer in Test Example 5 of this invention; Figure 8 This is a bar chart showing the incidence of ovarian cysts in each group of sika deer in Test Example 5 of this invention. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings, examples, comparative examples, and test examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Raw materials: Follicle-stimulating hormone (FSH), CAS No.: 9002-68-0; High molecular weight sodium hyaluronate, CAS No.: 9067-32-7; Hyaluronidase, CAS No.: 9001-54-1; Dextran T70, CAS No.: 9004-54-0; Trehalose, CAS No.: 6138-23-4; Zinc gluconate, CAS No.: 4468-02-4; Pregnant mare serum gonadotropin (PMSG), CAS No.: 9002-70-4; Progesterone, CAS No.: 57-83-0.

[0034] Preparation Examples 1-6: Preparation Example 1: This preparation example provides a method for preparing medium-to-low molecular weight sodium hyaluronate with a weight-average molecular weight of 10 kDa, including the following steps: High molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000 kDa to 1500 kDa was dissolved in phosphate buffer at pH 6.0 to prepare a 1% (w / w) solution. Hyaluronidase was added to the solution at a concentration of 5000 U / g substrate. The enzymatic hydrolysis reaction was carried out in a constant temperature water bath at 37°C for 6 hours. After the reaction was completed, the temperature was rapidly increased to 90°C and held for 10 minutes to inactivate the enzyme. The reaction solution was centrifuged and the supernatant was collected. Two-stage purification was performed using a tangential flow ultrafiltration system. First, the filtrate was collected through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa. Then, the filtrate was collected through an ultrafiltration membrane with a molecular weight cutoff of 5 kDa. The final retentate was dialyzed to remove salt and then freeze-dried. The sodium hyaluronate with a weight-average molecular weight of 10 kDa was obtained by gel permeation chromatography.

[0035] Preparation Example 2: This preparation example provides a method for preparing medium-to-low molecular weight sodium hyaluronate with a weight-average molecular weight of 30 kDa, including the following steps: High molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000 kDa to 1500 kDa was dissolved in phosphate buffer at pH 6.2 to prepare a 1% (w / w) solution. Hyaluronidase was added to the solution at a concentration of 3000 U / g substrate, and the enzymatic hydrolysis reaction was carried out in a constant temperature water bath at 37°C for 4 hours. After the reaction was completed, the temperature was rapidly increased to 90°C and kept at 90°C for 10 minutes to inactivate the enzyme. The reaction solution was centrifuged and the supernatant was collected. The solution was purified using a tangential flow ultrafiltration system. The filtrate was first collected through an ultrafiltration membrane with a molecular weight cutoff of 50 kDa, and then the filtrate was collected through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa. The final retentate was dialyzed to remove salt and then freeze-dried. The sodium hyaluronate with a weight-average molecular weight of 30 kDa was obtained by gel permeation chromatography.

[0036] Preparation Example 3: This preparation example provides a method for preparing medium-to-low molecular weight sodium hyaluronate with a weight-average molecular weight of 50 kDa, including the following steps: High molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000 kDa to 1500 kDa was dissolved in phosphate buffer at pH 6.5 to prepare a 1% (w / w) solution. Hyaluronidase was added to the solution at a concentration of 1000 U / g substrate. The enzymatic hydrolysis reaction was carried out in a constant temperature water bath at 37°C for 2 hours. After the reaction was completed, the temperature was rapidly increased to 90°C and kept at 90°C for 10 minutes to inactivate the enzyme. The reaction solution was centrifuged and the supernatant was collected. The solution was purified using a tangential flow ultrafiltration system. The filtrate was first collected through an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and then the filtrate was collected through an ultrafiltration membrane with a molecular weight cutoff of 30 kDa. The final retentate was dialyzed to remove salt and then freeze-dried. The hyaluronic acid with a weight-average molecular weight of 50 kDa was obtained by gel permeation chromatography.

[0037] Preparation Example 4: This preparation example provides a method for preparing low molecular weight dextran sulfate with a weight-average molecular weight of 36 kDa, including the following steps: Dextran T70 was placed in a 0.1 mol / L hydrochloric acid solution and subjected to controlled partial acid hydrolysis at 60 °C for 3 h. When the weight-average molecular weight dropped to approximately 18 kDa as monitored by gel permeation chromatography, the reaction was immediately terminated by neutralizing the pH to 7.0 with 1 mol / L sodium hydroxide solution. After precipitation with anhydrous ethanol and vacuum drying, a low molecular weight dextran intermediate was obtained. This intermediate was completely dissolved in N,N-dimethylformamide, and then an equal volume of anhydrous pyridine was added. A chlorosulfonic acid-pyridine complex (the mass ratio of chlorosulfonic acid to dextran intermediate was 2:1) was slowly added dropwise at 60 °C for sulfation modification for 4 h. After the reaction was completed, the mixture was dialyzed with deionized water for 48 h to remove free reagents. Finally, it was freeze-dried, and the result was analyzed by gel permeation chromatography to obtain sulfated dextran with a weight-average molecular weight of 36 kDa.

[0038] Preparation Example 5: This preparation example provides a method for preparing low molecular weight dextran sulfate with a weight-average molecular weight of 43 kDa, including the following steps: Dextran T70 was placed in a 0.1 mol / L hydrochloric acid solution and subjected to controlled partial acid hydrolysis at 60 °C for 2 h. When the weight-average molecular weight dropped to approximately 21 kDa as monitored by gel permeation chromatography, the reaction was immediately terminated by neutralizing the pH to 7.0 with a 1 mol / L sodium hydroxide solution. After precipitation with anhydrous ethanol and vacuum drying, a low molecular weight dextran intermediate was obtained. This intermediate was completely dissolved in N,N-dimethylformamide, and then an equal volume of anhydrous pyridine was added. A chlorosulfonic acid-pyridine complex (the mass ratio of chlorosulfonic acid to dextran intermediate was 2:1) was slowly added dropwise at 60 °C for sulfation modification. The reaction time was 3 h. After the reaction was completed, the mixture was dialyzed with deionized water for 48 h to remove free reagents. Finally, it was freeze-dried, and the result was analyzed by gel permeation chromatography to obtain sulfated dextran with a weight-average molecular weight of 43 kDa.

[0039] Preparation Example 6: This preparation example provides a method for preparing low molecular weight dextran sulfated sugar with a weight-average molecular weight of 50 kDa, including the following steps: Dextran T70 was placed in a 0.1 mol / L hydrochloric acid solution and subjected to controlled partial acid hydrolysis at 60 °C for 1 h. When the weight-average molecular weight dropped to approximately 25 kDa as monitored by gel permeation chromatography, the reaction was immediately terminated by neutralizing the pH to 7.0 with a 1 mol / L sodium hydroxide solution. After precipitation with anhydrous ethanol and vacuum drying, a low molecular weight dextran intermediate was obtained. This intermediate was completely dissolved in N,N-dimethylformamide, and then an equal volume of anhydrous pyridine was added. A chlorosulfonic acid-pyridine complex (the mass ratio of chlorosulfonic acid to dextran intermediate was 2:1) was slowly added dropwise at 60 °C for sulfation modification. The reaction time was 2 h. After the reaction was completed, the mixture was dialyzed with deionized water for 48 h to remove free reagents. Finally, it was freeze-dried, and the result was analyzed by gel permeation chromatography to obtain sulfated dextran with a weight-average molecular weight of 50 kDa.

[0040] Examples 1-3: Example 1

[0041] This embodiment provides a twin pregnancy regulator for sika deer males that synchronize estrus and its application method, including the following steps: Preparation of the shielding base solution: Measure 8 mL of 0.9% physiological saline in a sterile mixing tank and add 0.2 g of trehalose. After dissolving with magnetic stirring, add a trace amount of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer to precisely adjust the pH of the system to 7.2. Turn on the cooling circulation system to cool the base solution and maintain it at 2°C.

[0042] Initial dissolution of active peptides: Under constant temperature of 2℃, 50 IU of lyophilized pituitary follicle-stimulating hormone powder was added to the above-mentioned shielding base solution and stirred at 50 rpm until completely dissolved.

[0043] Polyelectrolyte hydration and steric hindrance locking: While maintaining a constant temperature of 2°C, 20 mg of dextran sulfate obtained in Preparation Example 4 and 100 mg of sodium hyaluronate obtained in Preparation Example 1 were slowly and evenly dispersed into the system in sequence, and the mixture was stirred at a low speed until the powders were completely hydrated and dissolved to form a homogeneous and transparent sol system.

[0044] Microscopic coordination and dispensing: The above sol was vortexed at 1000 rpm, and a pre-prepared zinc gluconate aqueous solution was slowly added dropwise to bring the final zinc ion concentration in the system to 0.5 mmol / L. The final volume of the system was adjusted to 10 mL with 0.9% physiological saline, and the mixture was continuously vortexed for 5 min. The solution was dispensed into sterile syringes at a rate of 2 mL / vial, sealed, and stored at 2°C in the dark for later use.

[0045] Twin pregnancy regulation application: Select healthy female sika deer, disinfect the vulva with 0.1% potassium permanganate solution, and implant a vaginal silicone plug containing progesterone deep into the vagina. The day of implantation is recorded as day 0. Remove the vaginal silicone plug on the 12th day after implantation. At the same time as plug removal, inject 330 IU of pregnant mare serum gonadotropin into the neck muscle of the female deer. Take out a syringe pre-filled with 2 mL of the above twin pregnancy regulation agent from a 2°C refrigerated environment and inject it into the deep muscle of the female deer's buttocks. 36 hours after plug removal and drug administration, introduce the female deer into the breeding pen and introduce a healthy male deer for natural mating. Example 2

[0046] This embodiment provides a twin pregnancy regulator for sika deer males that synchronize estrus and its application method, including the following steps: Preparation of shielding base solution: Measure 8 mL of 0.9% physiological saline in a sterile mixing tank, add 0.35 g of trehalose, stir magnetically to dissolve, and then add a trace amount of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer to precisely adjust the pH of the system to 7.3. Turn on the cooling circulation system to cool the base solution and keep it constant at 5°C.

[0047] Initial dissolution of active peptides: Under constant temperature of 5℃, 125 IU of lyophilized pituitary follicle-stimulating hormone powder was added to the above-mentioned shielding base solution and stirred at 100 rpm until completely dissolved.

[0048] Polyelectrolyte hydration and steric hindrance locking: While maintaining a constant temperature of 5°C, 60 mg of dextran sulfate obtained in Preparation Example 5 and 250 mg of sodium hyaluronate obtained in Preparation Example 2 were slowly and evenly dispersed into the system in sequence, and the mixture was stirred at a low speed until the powders were completely hydrated and dissolved to form a homogeneous and transparent sol system.

[0049] Microscopic coordination and dispensing: The above sol was vortexed at a frequency of 1750 rpm, and a pre-prepared zinc gluconate aqueous solution was slowly added dropwise to bring the final zinc ion concentration in the system to 1.5 mmol / L. The final volume of the system was adjusted to 10 mL with 0.9% physiological saline, and the mixture was continuously vortexed for 10 min. The solution was dispensed into sterile syringes at a rate of 2 mL / vial, sealed, and stored at 5°C in the dark for later use.

[0050] Twin pregnancy regulation application: Select healthy female sika deer, disinfect the vulva with 0.1% potassium permanganate solution, and implant a vaginal silicone plug containing progesterone deep into the vagina. The day of implantation is recorded as day 0. Remove the vaginal silicone plug on the 13th day after implantation. At the same time as plug removal, inject 340 IU of pregnant mare serum gonadotropin into the neck muscle of the female deer. Take out a syringe pre-filled with 2 mL of the above twin pregnancy regulation agent from a 5°C refrigerated environment and inject it into the deep muscle of the female deer's buttocks. Forty hours after plug removal and drug administration, introduce the female deer into the breeding pen and introduce a healthy male deer for natural mating. Example 3

[0051] This embodiment provides a twin pregnancy regulator for sika deer males that synchronize estrus and its application method, including the following steps: Preparation of shielding base solution: Measure 8 mL of 0.9% physiological saline in a sterile mixing tank, add 0.5 g of trehalose, stir magnetically to dissolve, and then add a trace amount of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer to precisely adjust the pH of the system to 7.4. Turn on the cooling circulation system to cool the base solution and keep it constant at 8°C.

[0052] Initial dissolution of active peptides: Under constant temperature of 8°C, 200 IU of lyophilized pituitary follicle-stimulating hormone powder was added to the above-mentioned shielding base solution and stirred at 150 rpm until completely dissolved.

[0053] Polyelectrolyte hydration and steric hindrance locking: While maintaining a constant temperature of 8°C, 100 mg of dextran sulfate obtained in Preparation Example 6 and 400 mg of sodium hyaluronate obtained in Preparation Example 3 were slowly and evenly dispersed into the system in sequence, and the mixture was stirred at a low speed until the powders were completely hydrated and dissolved to form a homogeneous and transparent sol system.

[0054] Microscopic coordination and dispensing: The above sol was vortexed at a frequency of 2500 rpm, and a pre-prepared zinc gluconate aqueous solution was slowly added dropwise to make the zinc ion concentration in the final system reach 2.5 mmol / L. The final volume of the system was adjusted to 10 mL with 0.9% physiological saline, and the mixture was continuously vortexed for 15 min. The solution was dispensed into sterile syringes at a rate of 2 mL / vial, sealed, and stored at 8°C in the dark for later use.

[0055] Twin pregnancy regulation application: Select healthy female sika deer, disinfect the vulva with 0.1% potassium permanganate solution, and implant a vaginal silicone plug containing progesterone deep into the vagina. The day of implantation is recorded as day 0. On the 14th day after implantation, remove the vaginal silicone plug. At the same time as plug removal, inject 350 IU of pregnant mare serum gonadotropin into the neck muscle of the female deer. Take out a syringe pre-filled with 2 mL of the above twin pregnancy regulation agent from the 8℃ refrigerated environment and inject it into the deep muscle of the female deer's buttocks. 44 hours after plug removal and drug administration, introduce the female deer into the breeding pen and introduce a healthy male deer for natural mating.

[0056] Comparative Examples 1-5: Comparative Example 1: Compared with Example 2, the difference is that dextran sulfate and sodium hyaluronate are not added in the polyelectrolyte hydration and steric hindrance locking steps, and zinc gluconate is not added in the micro-coordination and volume fixation and dispensing steps. Instead, 125 IU of pituitary follicle-stimulating hormone is directly dissolved in physiological saline containing trehalose to prepare a regular aqueous injection. All other steps are the same.

[0057] Comparative Example 2: Compared with Example 2, the difference is that trehalose is not added to the shielding base liquid in the preparation step of the shielding base liquid, but all other steps are the same.

[0058] Comparative Example 3: Compared with Example 2, the difference is that no zinc gluconate aqueous solution is added in the micro-coordination and volume adjustment and dispensing steps, that is, the system is completely lacking in divalent metal ions, while the rest are the same.

[0059] Comparative Example 4: Compared with Example 2, the difference is that only 250 mg of sodium hyaluronate obtained in Example 2 was added in the polyelectrolyte hydration and steric hindrance locking steps, and no dextran sulfate was added; all other steps were the same.

[0060] Comparative Example 5: Compared with Example 2, the difference is that the sodium hyaluronate in the polyelectrolyte hydration and steric hindrance locking steps is directly replaced by an equal mass of commercially available conventional high molecular weight sodium hyaluronate (initial weight-average molecular weight of 1000kDa to 1500kDa, without the enzymatic degradation treatment of Preparation Example 2), while all other steps are the same.

[0061] Test Examples 1-5: Test Example 1: This test example is used to determine the rheological characteristics of the drug solutions prepared in each embodiment and the comparative example under in vitro refrigeration environment, as well as the hydrogen bond shielding effect of trehalose. The specific experimental steps are as follows: The test temperature of the rotational rheometer was set to 5℃, a cone-plate measurement system was used, the cone diameter was set to 40mm, the cone angle was 2°, and the test gap was set to 0.05mm. Take 1.5 mL of each of the formulations from Example 2, Comparative Example 2, and Comparative Example 3, place them on the test platform of the rheometer, lower the measuring cone plate, and scrape off any sample residue that overflows from the edge. The sample was kept at a constant temperature of 5℃ for 3 minutes to eliminate the residual internal flow stress generated during the sample loading and closing process. Start the steady-state shear scan program and set the test interval for the shear rate to 0.1 s. -1 up to 100s -1 The test points were collected using a logarithmic distribution model, and the apparent viscosity values ​​of each group of samples were recorded at different shear rates. Each independent sample was measured three times and the arithmetic mean was taken.

[0062] Table 1. Apparent viscosity test results at different shear rates under 5℃. <![CDATA[Shear rate (s -1 ).]]> Example 2 Apparent viscosity (Pa·s) Comparative Example 2: Apparent viscosity (Pa·s) Comparative Example 3: Apparent viscosity (Pa·s) 0.1 0.94 85.62 0.81 0.5 0.61 51.27 0.54 1.0 0.48 32.84 0.42 5.0 0.23 14.19 0.20 10.0 0.17 8.63 0.14 50.0 0.09 3.88 0.08 100.0 0.06 2.14 0.05 Summary: Refer to the appendix Figure 1 and attached Figure 2 Based on the data in Table 1, under a test environment of 5℃, in Comparative Example 3, since the system does not contain divalent zinc ions, sodium hyaluronate and dextran sulfate mainly exist in the form of free polymeric segments, exhibiting the segmental entanglement characteristics of conventional polymeric aqueous solutions. This is particularly evident at low shear rates of 0.1 s⁻¹. -1 The apparent viscosity is 0.81 Pa·s, and it exhibits normal fluid rheological behavior with increasing shear rate.

[0063] In Comparative Example 2, after removing trehalose from the formulation, the apparent viscosity of the system increased sharply by orders of magnitude, reaching a maximum of 0.1 s⁻¹. -1 The viscosity reached 85.62 Pa·s. Due to the lack of physical isolation by trehalose, the carboxyl groups of sodium hyaluronate and the sulfate groups of dextran sulfate were directly exposed in the aqueous solution. They underwent coordination and cross-linking reactions with free divalent zinc ions in the system. The polymer chains were physically bridged by metal ions, which led to phase separation and gelation transformation of the drug solution in the low temperature environment in vitro, forming a high-viscosity network structure and losing the free flowability required as an injection solution.

[0064] The apparent viscosity curve of Example 2 as a function of shear rate basically coincides with the data of Comparative Example 3. After introducing a certain concentration of trehalose into the formulation system, the polyhydroxyl groups in the trehalose molecule form a hydrogen bond hydration layer with water molecules and the polyelectrolyte backbone at low temperature. This hydrogen bond network constructs steric hindrance around the molecular chains of sodium hyaluronate and dextran sulfate, physically blocking the coordination channels between zinc ions and the anionic groups of the polymer backbone side chains. Zinc ions maintain a free and dispersed state in the low-temperature and high-osmotic microenvironment, inhibiting early cross-linking between polymer chains, and keeping the macroscopic formulation in a low-viscosity sol state. This verifies the role of in vitro osmotic pressure blocking and hydrogen bond shielding mechanisms in maintaining the physical stability of the formulation and ensuring its clinical injectability.

[0065] Test Example 2: This test example is used to determine the osmotic pressure displacement rate and in-situ condensation phase transition characteristics of the drug solution prepared in the example under a simulated animal tissue fluid environment. The specific experimental steps are as follows: Cellulose dialysis bags with a molecular weight cutoff of 500 Da were boiled in boiling water for 10 minutes to remove the surface anti-corrosion coating, rinsed with deionized water, and then soaked in 0.9% sodium chloride solution for later use. Accurately pipette 2 mL of the drug solution prepared in Example 2, put it into the prepared dialysis bag, remove any residual air inside, and seal both ends with dialysis clamps; Immerse the dialysis bag containing the medication into a constant-temperature receiving bath containing 50 mL of 0.9% sodium chloride solution. Adjust the water bath temperature to 38.5°C, turn on the magnetic stirrer, and set the speed to 50 rpm. When the reaction system runs for 5 min, 15 min, 30 min, 60 min and 120 min, accurately remove 1 mL of release medium from the receiving cell. After each sampling, add 1 mL of 0.9% sodium chloride solution preheated to 38.5℃ to the receiving cell. The concentration of trehalose in the obtained medium was determined using a high-performance liquid chromatograph equipped with a differential refractive index detector. An amino column was used, and the mobile phase was a solution of acetonitrile and water at a volume ratio of 75:25. The cumulative percolation rate at each time point was calculated based on the standard curve, and the physical state of the drug solution inside the dialysis bag was observed and recorded at the corresponding points.

[0066] Table 2. Record of cumulative exudation rate and morphological changes of trehalose in simulated tissue fluid at 38.5℃ in Example 2 Time (min) Trehalose cumulative exudation rate (%) Macroscopic morphology of the drug solution inside the dialysis bag 0 0.0 Homogeneous transparent mobile sol 5 24.3 Localized slight turbidity, overall fluidity begins to decrease 15 51.8 It exhibits a semi-transparent, viscous, soft gel-like consistency and has lost its fluidity. 30 79.2 They aggregate into irregular gel blocks with clearly defined boundaries. 60 88.6 The gel block shrinks in volume and becomes denser. 120 93.1 Formation of compact, water-insoluble, hard phase change complexes Summary: Refer to the appendix Figure 3According to the data in Table 2, after the formulation of Example 2 was placed in a simulated physiological saline environment at 38.5°C with no external trehalose, a high osmotic pressure gradient was formed between the trehalose concentration inside and outside the dialysis bag. Driven by the thermodynamic gradient, trehalose diffused through the dialysis membrane into the hypotonic medium. The cumulative percolation rate reached 51.8% in the first 15 minutes and 88.6% in 60 minutes.

[0067] Simultaneously with the high-speed escape of trehalose, the physical state of the sodium hyaluronate and dextran sulfate macromolecules trapped inside the dialysis bag changes, collapsing from an initial liquid sol into a dense, water-insoluble aggregate. The high concentration of trehalose inside the drug solution diffuses into the surrounding medium, causing the hydrogen bond hydration shielding layer surrounding the polymer chain segments to disintegrate.

[0068] After the steric hindrance and hydrogen bond network are eliminated, the exposed polyvalent anionic groups on the macromolecular backbone and the free zinc ions in the system undergo a high-density multi-coordination bridging reaction in the locally confined space. The electrostatic repulsion between molecular chains is weakened, and hydrophobic interactions become dominant, which promotes the liquid-solid phase separation of the polymeric polyelectrolyte. The test results verify that after the formulation comes into contact with the living tissue fluid environment, it can spontaneously peel off the protective layer by relying on the microscopic osmotic pressure gradient, triggering the in-situ cross-linking of metal ions and polyanionic backbone, and self-assembling at the drug administration site to form a stable physical sustained-release backbone.

[0069] Test Example 3: This test example is used to determine the injection force and injectability of the drug solutions prepared in each embodiment and comparative example under low temperature conditions. The specific experimental steps are as follows: Select a standard 5mL veterinary polypropylene disposable syringe, with an 18G stainless steel injection needle connected to the front end; In a sterile cold chain operating table, 2 mL of each of the refrigerated preparation samples of Example 1, Example 2, Example 3, Comparative Example 2, and Comparative Example 5 were drawn into syringes and slowly injected to expel the air from the tube and needle. The syringe containing the drug sample is vertically fixed on the pressure test fixture of the universal material testing machine with a constant temperature control environment chamber. The temperature of the environment chamber is adjusted to be maintained at 5°C, and the sample is allowed to stand and balance in the fixture for 5 minutes. The downward displacement rate of the testing machine's pressure plate is set to a constant 1.0 mm / s. The test program is started, and load data is recorded after the downward pressure plate contacts the syringe piston rod. The force data of the piston pushing across its entire stroke is collected and output in real time by a mechanical sensor. The peak thrust of each test group is extracted, and three parallel samples are set for repeated testing in each group.

[0070] Table 3. Peak injection force test results for each formulation at a constant injection rate of 3.5℃

[0071] Summary: Refer to the appendix Figure 4 and attached Figure 5 According to the data in Table 3, the peak injection force of Examples 1, 2 and 3 is distributed in the range of 10.5N to 27.8N. Under the conditions of 5°C low temperature environment and the presence of trehalose, the thrust of Example 3 is maintained within the range of veterinary manual injection operation below 30N. The formulation exhibits low shear resistance fluid characteristics in the narrow channel of the syringe, avoiding stress behavior in the doe at the breeding site.

[0072] Comparative Example 2, without the addition of trehalose for hydrogen bond shielding, showed that zinc ions coordinated with the anionic groups of dextran sulfate and sodium hyaluronate during solution preparation and refrigeration, resulting in increased system viscosity and a peak thrust of 138.2 N to 152.1 N, exceeding the physiological force limit for single-handed injection. Comparative Example 5 used high molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000 kDa to 1500 kDa, replacing the low molecular weight fragment of 30 kDa specified in this scheme. Its peak thrust increased to 79.5 N to 86.9 N. The molecular chains of high molecular weight polysaccharides exhibited dense chain entanglement in the solution, generating higher flow resistance.

[0073] This invention solves the engineering application problem of excessively high viscosity in the drug delivery stage of a polymeric polyelectrolyte cross-linked sustained-release system by selecting a sodium hyaluronate backbone with a specific degraded molecular weight, combined with trehalose microosmotic pressure and hydrogen bond blocking technology.

[0074] Test Example 4: This test example is used to determine the kinetic characteristics of follicle-stimulating hormone release and the sustained-release locking effect of the drug solutions prepared in each example and comparative example after simulated phase transition in vitro. The specific experimental steps are as follows: Phosphate-buffered saline solution with a pH of 7.4 was prepared as the in vitro release medium. The medium was preheated in a constant temperature water bath and maintained at 38.5°C. Using a membrane-free in-situ release model, a 50 mL stoppered glass test tube was used, and 10 mL of preheated release medium was added to it. Using a sterile syringe, 2 mL of the drug solution from Examples 2, 1, 3, and 4, which had been pre-refrigerated at 5°C, was drawn. The syringe needle was inserted into the bottom of the test tube, and the drug solution was slowly injected into the release medium. The initial phase transition state of the drug solution after contact with the high-temperature medium was recorded. Fix all test tubes in a constant temperature shaking water bath, set the water bath temperature to 38.5℃ and the shaking frequency to 50rpm; At 2h, 12h, 24h, 48h, 72h and 96h of the experiment, 1mL of release medium was accurately transferred from each test tube for concentration detection. After each sampling, 1mL of fresh release medium at 38.5℃ was added to the test tube. The optical density of the sampled medium at each time point was measured using a follicle-stimulating hormone (FSH) enzyme-linked immunosorbent assay (ELISA) kit at a wavelength of 450 nm. The mass concentration of free pituitary FSH in the medium was calculated by combining the standard curve and converted into the cumulative release rate.

[0075] Table 4. Results of cumulative release rate of pituitary follicle-stimulating hormone in each group in the in vitro release model at 38.5℃

[0076] Summary: Refer to the appendix Figure 6 According to the data in Table 4, Comparative Example 1, as a conventional aqueous injection without the addition of polymer backbone and cross-linking agent, showed a cumulative release rate of 88.4% within 2 hours after injection into the release medium at 38.5℃, exhibiting a burst release effect. The pituitary follicle-stimulating hormone molecules diffused freely into the surrounding medium under the drive of the concentration gradient, which would cause fluctuations in blood drug concentration in in vivo applications, deviating from the physiological threshold for stable follicle development.

[0077] Comparative Example 3 removed divalent zinc ions from the formulation, resulting in a lack of metal coordination crosslinking centers in the system. The linear polymer could only provide limited physical viscous resistance in the medium, and the drug solution could not undergo a condensation phase transition from liquid to solid after contacting the medium. Its release rate reached 74.2% after 12 hours, failing to form a sustained-release reservoir. Comparative Example 4 did not add dextran sulfate to the polyelectrolyte skeleton, retaining only a single sodium hyaluronate network. The coordination ability of the carboxyl groups on the sodium hyaluronate molecular chain with zinc ions was weaker than that of the sulfate groups, resulting in a lower density of local crosslinking nodes and a larger pore size in the gel network. This led to an initial release rate of 28.2% after 2 hours and more than half of the drug was released after 12 hours, indicating an incomplete drug locking effect.

[0078] In Example 2, after injection of a high-temperature, low-osmotic medium, trehalose diffused outwards and emptied, exposing the carboxyl groups of sodium hyaluronate and the sulfate groups of dextran sulfate, which then formed multiple ion bridges with zinc ions in situ. The system self-assembled and collapsed to form an ion-aggregated complex, trapping pituitary follicle-stimulating hormone within the cross-linked network. In Example 2, the initial release rate was 8.6% after 2 hours, suppressing the risk of burst release. During the subsequent 96-hour test period, the drug was released with near-zero-order kinetics, with a release rate of 77.2% at 72 hours and 92.5% at 96 hours. The test data confirmed that the in-situ osmotic pressure displacement and multivalent anion co-aggregation mechanism can construct a stable drug delivery microenvironment, allowing the release rate of reproductive hormones to match the physiological time window of double ovulation in female deer.

[0079] Test Example 5: This test case was used to determine the twin-pregnancy regulation effect and reproductive endocrine safety of the drug solutions prepared in the examples and comparative examples in a live estrus synchronization experiment of sika deer. The specific experimental steps are as follows: Two hundred and fifty multiparous female sika deer of similar weight and without reproductive tract diseases, who were in the breeding season, were selected and randomly divided into five experimental groups. Example 1 group had 50 deer, Example 2 group had 50 deer, Example 3 group had 50 deer, Comparative Example 1 group had 50 deer, and Comparative Example 4 group had 50 deer.

[0080] The vulva of each group of does was cleaned and disinfected using a 0.1% potassium permanganate solution, and progesterone-containing vaginal suppositories were inserted deep into the vagina of the does.

[0081] According to the medication time points set for each group, the vaginal plugs were removed, and at the same time as the plugs were removed, the corresponding dose of pregnant mare serum gonadotropin was injected into the neck muscle of the doe.

[0082] Two mL of the refrigerated drug solution from Examples 1 to 3, Comparative Example 1 and Comparative Example 4 were extracted and injected into the deep muscle of the buttocks of the doe.

[0083] At the corresponding time points after drug administration, the female deer in each group were introduced into the mating enclosure and placed in healthy male deer that had passed the semen motility test for natural mating.

[0084] After mating, the estrus behavior of the female deer in each group was observed and recorded, and the estrus rate during the same period was calculated.

[0085] On the 45th day after the end of this mating, a veterinary ultrasound was used to perform a rectal ultrasound pregnancy examination on the does, to record the pregnancy status, and to check and record whether there were any anovulatory cystic structures in the ovaries.

[0086] Record the birthing data of does in the following year's calving season and calculate the proportion of does that gave birth to twins out of the total number of pregnant does.

[0087] Table 5. Statistical data on the effects of reproductive physiological regulation on does in each group.

[0088] Summary: Refer to the appendix Figure 7 and attached Figure 8 Based on the data in Table 5, Comparative Example 1, serving as the control group for conventional pituitary follicle-stimulating hormone (FSH) aqueous injection, showed a estrus synchronization rate of 92.0% and a twin pregnancy rate of only 2.4%, essentially the same as the twin pregnancy rate under natural mating conditions. Furthermore, the incidence of ovarian cysts reached 20.0%. The aqueous injection lacks diffusion resistance after intramuscular injection, causing FSH to rapidly dissolve in local tissue fluid and enter the bloodstream, resulting in a momentary peak in blood drug concentration. This gonadotropin concentration exceeding the physiological tolerance threshold triggers ovarian hyperstimulation, leading to atresia cysts in some follicles after reaching the dominant stage of development, disrupting the endocrine environment for the co-development of two dominant follicles.

[0089] Comparative Example 4 used only a single sodium hyaluronate network, lacking the synergistic cross-linking of dextran sulfate. The resulting gel reservoir structure had limited ability to retain reproductive hormones, and the twin pregnancy rate increased to 9.5%, but the ovarian cyst rate was still 12.0%.

[0090] The measurement data from Examples 1 to 3 show that after using the twin pregnancy regulator of the present invention, the estrus synchronization rate of does remained stable at approximately 94.0% to 96.0%, the twin pregnancy rate significantly increased to nearly 25% (range 22.7% to 26.7%), and the incidence of ovarian cysts was controlled at a low level of 2.0% to 4.0%. After the drug solution is injected into the intermuscular space, the high concentration of trehalose molecules within the system diffuses into the surrounding tissue fluid under the drive of the local osmotic pressure gradient. Trehalose stripping leads to the disintegration of the hydrogen bond hydration barrier layer on the surface of the polysaccharide macromolecules. After the loss of steric hindrance, the exposed carboxyl and sulfate groups undergo multiple ion bridging with free zinc ions in the narrow space of the muscle.

[0091] The system consists of a low-viscosity fluid that collapses and self-assembles in situ to form a highly viscoelastic polyelectrolyte condensate. This condensate acts as a physical insulating framework, reducing the mass transfer and diffusion coefficient of the encapsulated follicle-stimulating hormone (FSH). As enzymes in the animal's body degrade the polysaccharide framework, FSH is released into the bloodstream at a uniform rate, maintaining the blood drug concentration within the therapeutic window that promotes the simultaneous development of two dominant follicles. Zinc ions are simultaneously released into the local microcirculation during the framework disintegration process, participating in oocyte metabolism and achieving a stable increase in the efficiency of synchronized estrus mating and twinning rates in sika deer.

Claims

1. A twin pregnancy regulator for sika deer males in the same mating species, characterized in that, The twin-fetal regulator is a homogeneous and transparent sol system; The twin pregnancy regulator is made from the following raw material components in the following proportions: Each 8-12 mL system contains: 200-500mg trehalose; 50-200 IU of pituitary follicle-stimulating hormone; 100-400mg of medium to low molecular weight sodium hyaluronate; 20-100mg low molecular weight dextran sulfate; 1.8-14 mg zinc gluconate; Furthermore, the twin pregnancy regulator is diluted to volume with physiological saline at a mass concentration of 0.8%-1.0%, and the pH value of the twin pregnancy regulator is 7.2-7.4; The sodium hyaluronate of medium and low molecular weight has a weight-average molecular weight of 10kDa-50kDa, the dextran sulfate of low molecular weight has a weight-average molecular weight of 36kDa-50kDa, and the zinc gluconate provides a final zinc ion concentration of 0.5-2.5 mmol / L in the system.

2. The twin pregnancy regulator for sika deer males with synchronized estrus according to claim 1, characterized in that, In the raw material components of the twin pregnancy regulator, the mass ratio of the medium-low molecular weight sodium hyaluronate to the low molecular weight dextran sulfate is 3.0-4.5:1, and the mass ratio of the trehalose to the medium-low molecular weight sodium hyaluronate is 1.0-1.5:

1.

3. The twin pregnancy regulator for sika deer males with synchronized estrus according to claim 1, characterized in that, The medium- and low-molecular-weight sodium hyaluronate is prepared by the following steps: Take high molecular weight sodium hyaluronate with an initial weight-average molecular weight of 1000kDa-1500kDa, dissolve it in phosphate buffer solution with a pH of 6.0-6.5, and prepare a solution with a mass fraction of 0.8%-1.2%. Add hyaluronidase at a dosage of 1000-5000 U / g of substrate to the solution, and carry out the enzymatic hydrolysis reaction in a constant temperature water bath at 35-40℃ for 2-6 hours to obtain the reaction solution; The reaction solution is heated to 85-95℃ and kept at that temperature for 8-15 minutes to inactivate the enzyme, resulting in an inactivated reaction solution. The inactivated reaction solution is then centrifuged to collect the supernatant. The supernatant is then purified in two stages using a tangential flow ultrafiltration system. First, the primary filtrate is collected through an ultrafiltration membrane with a molecular weight cutoff of 50-100 kDa. Then, the primary filtrate is collected through an ultrafiltration membrane with a molecular weight cutoff of 5-30 kDa. After dialysis to remove salt from the retentate, the solution is freeze-dried to obtain the medium-low molecular weight sodium hyaluronate.

4. The twin pregnancy regulator for sika deer males with synchronized estrus according to claim 1, characterized in that, The low molecular weight dextran sulfated sugar is prepared by the following steps: Dextran T70 was placed in a 0.08-0.12 mol / L hydrochloric acid solution and subjected to acid hydrolysis at 55-65℃ for 1-3 hours. When the weight-average molecular weight decreased to 18-25 kDa, the reaction was terminated by neutralizing with a 0.8-1.2 mol / L sodium hydroxide solution to a pH of 6.8-7.2, yielding a reaction termination solution. The reaction termination solution was then precipitated with anhydrous ethanol and vacuum dried to obtain a low molecular weight dextran intermediate. The low molecular weight dextran intermediate was dissolved in N,N-dimethylformamide to form a solution. Then, anhydrous pyridine was added to the solution at a volume ratio of 0.8-1.2:

1. The solution was then sulfated by dropwise addition of a chlorosulfonic acid-pyridine complex at 55-65°C for 2-4 hours to obtain a modified solution. The mass ratio of chlorosulfonic acid to the low molecular weight dextran intermediate was 1.8-2.2:

1. The modified solution was dialyzed with deionized water for 40-50 hours, and then freeze-dried to obtain the low molecular weight dextran sulfate.

5. The twin pregnancy regulator for sika deer males with synchronized estrus according to claim 1, characterized in that, The twin pregnancy regulator is prepared by the following steps: Measure 7-9 mL of physiological saline with a mass concentration of 0.8%-1.0% into a sterile preparation tank, add trehalose, and dissolve it by magnetic stirring to obtain a trehalose initial solution. Add sodium dihydrogen phosphate-disodium hydrogen phosphate buffer dropwise to the trehalose initial solution to adjust the pH of the system to 7.2-7.4 to obtain the base solution. Turn on the cooling circulation system to cool the base solution and keep it constant at 2℃-8℃. Under constant temperature conditions of 2℃-8℃, the lyophilized pituitary follicle-stimulating hormone powder was added to the base solution and stirred to dissolve, thus obtaining a preliminary solution system. While maintaining a constant temperature, the low molecular weight dextran sulfate and the medium-low molecular weight sodium hyaluronate are sequentially dispersed into the initial dissolution system, and the mixture is continuously stirred at a low speed until the powder dissolves to form a sol system. The sol system was vortexed, and a pre-prepared zinc gluconate aqueous solution was added dropwise during the process to bring the zinc ion concentration in the system to 0.5-2.5 mmol / L, thus obtaining a zinc-containing sol. The zinc-containing sol was then brought to a final volume of 8-12 mL with physiological saline with a mass concentration of 0.8%-1.0%, and the mixture was continuously vortexed to obtain a homogeneous regulator sol. The homogeneous regulator sol was dispensed into sterile syringes, sealed, and stored at 2℃-8℃ in the dark for later use, thus obtaining the twin pregnancy regulator.

6. The twin pregnancy regulator for sika deer males with synchronized estrus according to claim 5, characterized in that, During the process of adding the lyophilized pituitary follicle-stimulating hormone powder to the base solution and stirring to dissolve it, the stirring speed is controlled at 50-150 rpm. During the process of oscillating the sol system using a vortex oscillator, the oscillation frequency of the vortex oscillator is 1000-2500 rpm, and the continuous oscillation and mixing time is 5-15 min.

7. A method for applying the twin pregnancy regulator for sika deer synchronous estrus males as described in any one of claims 1-6, characterized in that, Includes the following steps: A vaginal silicone plug containing progesterone was implanted deep into the vagina of a healthy female sika deer, and the day of implantation was recorded as day 0. The vaginal silicone plug was removed 12-14 days after implantation. At the same time as plug removal, pregnant mare serum gonadotropin and the twin pregnancy regulator were injected into the muscle area of ​​the mother sika deer. 36-44 hours after the thrombectomy and drug administration, the female sika deer that has been injected with the twin pregnancy regulator is placed in the breeding pen, and a healthy male deer is introduced for natural mating.

8. The method for applying the twin pregnancy regulator for sika deer synchronous estrus males according to claim 7, characterized in that, Before implanting the vaginal silicone plug, the vulva of the female sika deer was disinfected and pretreated with a 0.08%-0.15% potassium permanganate solution.

9. The method for applying the twin pregnancy regulator for sika deer estrus-synchronizing males according to claim 7, characterized in that, The pregnant mare serum gonadotropin was injected into the neck muscles of the female sika deer at a dose of 330-350 IU.

10. The method for applying the twin pregnancy regulator for sika deer synchronous estrus males according to claim 7, characterized in that, The twin pregnancy regulator is injected into the deep muscle of the buttocks of the female sika deer, with an injection dosage of 1.5-2.5 mL / vial.

Images