Pharmaceutical combination for improving reproductive and production performance of dam

Abstract

The present invention relates to the field of fusion proteins. Disclosed is a pharmaceutical combination for improving the reproductive and production performance of a dam. Specifically, provided in the present invention are a pharmaceutical composition, a kit or a pharmaceutical kit for improving the reproductive and production performance of a dam. More specifically, the pharmaceutical composition contains long-acting FSH and a luteinizing hormone drug. The present invention further relates to a method and the use for improving the reproductive performance of a dam by means of the combined use of long-acting FSH and a luteinizing hormone drug.

Description

A combination of drugs to improve the reproductive performance of female livestock

[0001] Cross-references to related applications

[0002] This application claims the benefit of Chinese Patent Application No. 202510017768.8, filed on January 6, 2025, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of veterinary drugs, and in particular to drugs that improve the reproductive and productive performance of female livestock. Background Technology

[0004] Reproductive regulation in mammals is primarily controlled by the hypothalamic-pituitary-gonadal axis. The hypothalamus secretes GnRH, which acts on the pituitary gland to stimulate the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH act on the ovaries to promote follicle development and ovulation, and also promote the synthesis and secretion of hormones. The most important protein hormones in the process of promoting follicle development and ovulation during reproduction are mainly divided into two categories: one is FSH-like hormones, which promote follicle development, including FSH and PMSG; the other is LH-like hormones, which promote ovulation, including LH and human chorionic gonadotropin (hCG).

[0005] Reproductive hormones are currently widely used in livestock production. Veterinary protein hormone products used for livestock reproductive regulation are mainly extracts. For example, FSH extracted from the porcine pituitary gland is primarily used for superovulation in cattle and sheep; PMSG extracted from pregnant mare serum is used for batch production in pigs and estrus synchronization in sheep; and hCG extracted from pregnant women's urine is used for ovulation induction. While these methods effectively improve production performance, the application of extracted hormones is limited by issues such as restricted raw material sources, high risk of disease transmission, and batch instability. Therefore, there is an urgent need for transformative extract-based hormone products in reproductive production to meet the growing demand for hormone products from livestock enterprises.

[0006] Recombinant protein hormones are obtained through cell expression and purification. They offer advantages such as high expression levels, high activity, low cost, batch stability, and yield not being limited by extraction processes and materials, effectively addressing the problems associated with extraction-based products. Based on the characteristics of female animal reproductive endocrine function and the types of hormones required for reproductive production, key protein products are developed. Due to the short half-life of wild-type FSH, its widespread application in animal reproduction is limited. Therefore, long-acting recombinant long-acting FSH protein products are developed using long-acting techniques to promote follicle development and are used for estrus synchronization and batch production in livestock. However, using recombinant FSH alone can lead to poor estrus and conception rates, reducing reproductive efficiency. In the area of ​​ovulation induction, recombinant hCG is developed, with its primary physiological function being ovulation promotion. However, even with attempts to promote ovulation, it is ineffective in improving reproductive efficiency when estrus and conception rates are poor. Overall, recombinant protein hormones offer high purity, high activity, batch stability, no potential sources of infection, and low cost, meeting the needs of veterinary drugs.

[0007] In response to the current state of the industry and the shortcomings and deficiencies of existing technologies, the inventors have creatively discovered that the combined use of FSH-type follicle-stimulating hormone drugs and LH-type drugs can significantly improve the estrus and conception effects of female animals, significantly improve reproductive productivity, and also provide a means of treating reproductive system-related diseases in female animals. Summary of the Invention

[0008] In response to the shortcomings of existing technologies and the current state of the industry, as well as the defects and deficiencies of existing technologies, the inventors have creatively discovered that the combined use of FSH-type follicle-stimulating hormone drugs and LH-type hormone drugs can significantly improve the estrus and conception effects of female animals, significantly improve reproductive productivity, and also provide a means of treating reproductive system-related diseases in female animals.

[0009] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows.

[0010] First aspect: drug combination

[0011] In one aspect of the invention, a pharmaceutical composition for improving the reproductive performance of female livestock is provided, which can be provided as a composition or a kit, and comprises a first substance and a second substance. The first substance is a long-acting FSH fusion protein, and the second substance can be luteinizing hormone (LH), long-acting luteinizing hormone, or human chorionic gonadotropin (hCG) and its analogues or functional equivalents.

[0012] In some specific implementation schemes, the first substance is: a long-acting FSH fusion protein, which contains FSH and a long-acting fragment, and the FSH portion and the long-acting fragment are linked by a linker or not; for example, it can be an FSH-Fc fusion protein or an FSH-CTP fusion protein, or an Fc-FSH fusion protein, or a CTP-FSH fusion protein, etc.; the linker protein can be a linker known in the prior art that does not affect the function of the functional element.

[0013] In some more specific embodiments, the first substance is a long-acting recombinant porcine FSH fusion protein, including, for example, fusion proteins pFSH-Fc-1 and pFSH-Fc-2, wherein the α subunit of the porcine FSH fusion protein pFSH-Fc-1 is fused directly or indirectly to the Fc fragment via a linker element, and the β subunit is bound to the α subunit via van der Waals forces or a linker element; the β subunit of the porcine FSH fusion protein pFSH-Fc-2 is fused directly or indirectly to the Fc fragment via a linker element, and the α subunit is bound to the β subunit via van der Waals forces or a linker element. The porcine FSH fusion protein comprises two peptide chains conforming to the following equation: (pFSHβ:pFSHα-L-Fc)2 or (pFSHα:pFSHβ-L-Fc)2, where pFSHβ refers to the β subunit of porcine FSH without the signal peptide; the colon represents the relationship between the porcine FSH β subunit and α subunit connected by van der Waals forces; pFSHα refers to the α subunit of porcine FSH without the signal peptide; L represents the connection relationship between the pFSHα or pFSHβ subunit and the Fc fragment; Fc refers to the Fc fragment of immunoglobulin or its mutant; the subscript 2 outside the parentheses indicates that the porcine FSH fusion protein is a divalent homodimer.

[0014] In some preferred embodiments, the amino acid sequence of pFSHα is as shown in SEQ ID NO:1, or a protein composed of an amino acid sequence that is more than 90% homologous to SEQ ID NO:1 and has the same function. The amino acid sequence of pFSHβ is as shown in SEQ ID NO:3, or a protein composed of an amino acid sequence that is more than 90% homologous to SEQ ID NO:3 and has the same function.

[0015] Additionally, the Fc may include an immunoglobulin hinge region as well as CH2 and CH3 regions. The immunoglobulins are derived from humans, pigs, cattle, sheep, horses, or dogs. Immunoglobulins are classified into IgG, IgM, IgA, IgD, and IgE, and each immunoglobulin includes various subtypes, such as IgG1, IgG2, IgG3, and IgG4.

[0016] The Fc mutant refers to an Fc variant containing one or more mutated amino acid sites on the Fc fragment, such as the human IgG2 Fc variant, which contains the Pro331Ser mutated human IgG2 hinge region, CH2 and CH3 region.

[0017] Preferably, the Fc is derived from porcine immunoglobulin, i.e., pFc, and includes the hinge region, CH2 region, and CH3 region of porcine immunoglobulin. The amino acid sequence of the pFc is as shown in SEQ ID NO:5, or is a protein composed of an amino acid sequence that has more than 80% homology with SEQ ID NO:5 and has the same function.

[0018] The connection between the pFSHα or pFSHβ subunit and Fc is either direct splicing or via a linker, preferably via a linker. The linker is a flexible polypeptide composed of 2-20 flexible amino acids, selected from at least one of Gly, Ser, Ala, and Thr. Preferably, the linker is (Gly-Gly-Gly-Gly-Ser). n , where n is an integer between 2 and 5, and more preferably n is 3.

[0019] Preferably, the pFSHα-L-Fc is:

[0020] i) A protein consisting of the amino acid sequence shown in SEQ ID No. 6; or

[0021] ii) Proteins derived from i) whose amino acid sequence shown in SEQ ID No. 6 has been substituted, deleted, and / or had one or more amino acids added, and which have the same function; or

[0022] iii) Proteins composed of amino acid sequences that are more than 90% homologous to the amino acid sequence shown in SEQ ID No. 6 and have the same function.

[0023] Preferably, the pFSHβ-L-Fc is:

[0024] iv) A protein consisting of the amino acid sequence shown in SEQ ID No. 8; or

[0025] v) Proteins derived from iv) by substitution, deletion, and / or addition of one or more amino acids of the amino acid sequence shown in SEQ ID No. 8, with equivalent function; or

[0026] vi) A protein consisting of an amino acid sequence that is more than 90% homologous to the amino acid sequence shown in SEQ ID No. 8 and has the same function.

[0027] In some specific embodiments, the first substance may be a recombinant porcine FSH-CTP fusion protein, wherein the fusion protein refers to a protein in which the β subunit of porcine FSH is directly or indirectly linked to the carboxyl-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin (hCG), and the α subunit of porcine FSH binds to the β subunit of porcine FSH via van der Waals forces. The hCG may be derived from humans, primates, or equines. Preferably, it is derived from humans or horses. Human CTP consists of 28 amino acid residues and contains 4 O-linked glycosylation sites; equine CTP consists of 35 amino acid residues and contains 12 O-linked glycosylation sites.

[0028] In some more specific embodiments, the fusion protein is either pFSH-hCTP or pFSH-eCTP.

[0029] pFSH-hCTP: Contains two peptide chains, conforming to the following equation: (pFSHα:pFSHβ-hCTP)2, where pFSHα refers to the α subunit of porcine FSH; the colon indicates that the porcine FSH α and β subunits are linked by van der Waals forces; pFSHβ refers to the β subunit of porcine FSH; hCTP refers to the carboxyl-terminal peptide of the β subunit of human chorionic gonadotropin; the subscript 2 outside the parentheses indicates that the porcine FSH fusion protein is a divalent homodimer.

[0030] Preferably, the pFSHβ-hCTP in the fusion protein pFSH-hCTP is:

[0031] i) A protein consisting of the amino acid sequence shown in SEQ ID NO:12; or

[0032] ii) Proteins derived from i) whose amino acid sequence shown in SEQ ID NO:12 has been substituted, deleted, and / or had one or more amino acids added, and which have the same function; or

[0033] iii) Proteins composed of amino acid sequences that are more than 90% homologous to the amino acid sequence shown in SEQ ID NO:12 and have the same function.

[0034] pFSH-eCTP: Contains two peptide chains, conforming to the following equation: (pFSHα:pFSHβ-eCTP)2, where pFSHα refers to the α subunit of porcine FSH; the colon indicates that the porcine FSH α and β subunits are linked by van der Waals forces; pFSHβ refers to the β subunit of porcine FSH; eCTP refers to the carboxyl-terminal peptide of the β subunit of equine chorionic gonadotropin; the subscript 2 outside the parentheses indicates that the porcine FSH fusion protein is a divalent homodimer.

[0035] Preferably, the pFSHβ-eCTP in the fusion protein pFSH-eCTP is:

[0036] iv) A protein consisting of the amino acid sequence shown in SEQ ID NO:14; or

[0037] v) Proteins derived from iv) by substituting, deleting, and / or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO:14, with equivalent function; or

[0038] vi) A protein consisting of an amino acid sequence that is more than 90% homologous to the amino acid sequence shown in SEQ ID NO:14 and has the same function.

[0039] The amino acid sequence of the pFSHα is as shown in SEQ ID NO:10, or a protein consisting of an amino acid sequence that is more than 90% homologous to SEQ ID NO:10 and has the same function.

[0040] It should also be noted that modified proteins, including two fusion proteins pFSH-hCTP, pFSH-eCTP, or porcine FSH that have undergone glycosylation, polyethylene glycolation, acetylation, or binding to BSA, are all within the scope of protection of this invention. Modified proteins, including two fusion proteins pFSH-hCTP, pFSH-eCTP, or fusion proteins formed by fusing porcine FSH protein with CTP or other proteins without altering the activity of porcine FSH protein, are all within the scope of protection of this invention.

[0041] In some specific embodiments, the second substance may be luteinizing hormone (LH), or long-acting luteinizing hormone, or hCG and its analogues or functional equivalents. For example, the long-acting luteinizing hormone may be LH-CTP, or CTP-LH, or LH-Fc, or Fc-LH, etc.; the hCG and its analogues or functional equivalents may be naturally derived hCG or hCG recombinantly expressed through bioengineering technology, or any other commercially available hCG, as long as it possesses the general biological functions of hCG. As examples only, hCG analogues may be goserelin, gonadorelin, etc.; hCG functional equivalents may be, for example, long-acting human chorionic gonadotropin (such as hCG-Fc), etc. In some preferred embodiments, the hCG is naturally derived hCG or hCG recombinantly expressed through bioengineering technology. In some more preferred embodiments, the hCG is recombinant hCG.

[0042] In some more specific embodiments, the second substance may be naturally derived hCG, such as an hCG product extracted from the urine of a pregnant woman. In other embodiments, the second substance may be recombinantly expressed hCG using bioengineering techniques, such recombinant hCG having the same sequence or structure as naturally derived hCG, for example, based on the gene sequences of the hCG α and β subunits published by NCBI. For example, such a recombinant hCG protein contains α and β subunits linked by van der Waals forces.

[0043] Wherein, the α subunit:

[0044] i) A protein consisting of the amino acid sequence shown in SEQ ID NO:16; or

[0045] ii) Proteins derived from i) by substitution, deletion, and / or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO:16, with equivalent function; or

[0046] iii) Proteins composed of amino acid sequences that are more than 90% homologous to the amino acid sequence shown in SEQ ID NO:16 and have the same function.

[0047] The β subunit:

[0048] i) A protein consisting of the amino acid sequence shown in SEQ ID NO:18; or

[0049] ii) Proteins derived from i) by substitution, deletion, and / or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO:18, with equivalent function; or

[0050] iii) Proteins composed of amino acid sequences that are more than 90% homologous to the amino acid sequence shown in SEQ ID NO:18 and have the same function.

[0051] The second aspect includes a kit or medicine box containing the composition.

[0052] In another aspect of the invention, a kit or pharmaceutical kit comprising the aforementioned drug combination is provided; or a kit or pharmaceutical kit comprising a first substance and a second substance packaged separately is provided.

[0053] In some embodiments, when the drug combination is provided as a composition or kit, the composition or kit may also contain necessary excipients, including but not limited to:

[0054] For kits or medicine boxes containing a specific potency of long-acting FSH, they may contain a prescription drug solution, such as 10-50 mM PB, 0.1-1% mannitol, 1-3% sucrose, 0.001-0.05% polysorbate 80, etc. The long-acting FSH can be formulated into a specific concentration (μg or U / ml) according to actual needs; alternatively, such long-acting FSH drug formulations can be provided as powder injections through freeze-drying.

[0055] For kits or pharmacies containing a specific potency of hCG, a prescription drug solution may be included, comprising 10-50 mM PB, 0.1-1% mannitol, 1-3% sucrose, and 0.001-0.05% polysorbate 80. Similarly, such hCG drug formulations can be provided as powder for injection by freeze-drying.

[0056] Alternatively, the pharmaceutical combination of the present invention can also be formulated as a pharmaceutical mixture containing a certain potency of FSH and hCG, contained in a kit or pharmaceutical composition, such a mixture may contain, for example, 30-50 μg of long-acting FSH and 200-400 IU of hCG; and a prescription solution comprising 10-50 mM PB, 0.1-1% mannitol, 1-3% sucrose, and 0.001-0.05% polysorbate 80. Similarly, such pharmaceutical mixture formulations can be provided as powder injections by freeze-drying.

[0057] In some more specific embodiments, the drug combination in the kit or reagent kit of the present invention may contain 30-50 μg of long-acting FSH, such as 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 μg of long-acting FSH. Simultaneously, such a kit or reagent kit may contain 200-400 IU of long-acting luteinizing hormone, hCG, or their analogues or functional equivalents, such as 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 IU of long-acting luteinizing hormone, hCG, or their analogues or functional equivalents, particularly hCG. The above-mentioned components can be prepared and administered in any combination of dosages within the corresponding range. For example, the drug combination may contain 30ug of long-acting FSH and 200IU of hCG, 35ug of long-acting FSH and 200IU of hCG, 40ug of long-acting FSH and 200IU of hCG, 45ug of long-acting FSH and 200IU of hCG, 50ug of long-acting FSH and 200IU of hCG, 30ug of long-acting FSH and 300IU of hCG, 35ug of long-acting FSH and 300IU of hCG, 40ug of long-acting FSH and 300IU of hCG, 45ug of long-acting FSH and 300IU of hCG, 5 0ug long-acting FSH and 300IU hCG, 30ug long-acting FSH and 400IU hCG, 35ug long-acting FSH and 400IU hCG, 40ug long-acting FSH and 400IU hCG, 45ug long-acting FSH and 400IU hCG, 50ug long-acting FSH and 400IU hCG, 30ug long-acting FSH and 500IU hCG, 35ug long-acting FSH and 500IU hCG, 40ug long-acting FSH and 500IU hCG, 45ug long-acting FSH and 500IU hCG or 50ug long-acting FSH and 500IU hCG.

[0058] The kit may further include an instruction manual that describes the components, uses, methods of use, dosage, and timing of administration of the drug combination, as well as other relevant information.

[0059] Third aspect: uses and methods of use

[0060] In one aspect of the invention, the use of a pharmaceutical combination comprising a first substance and a second substance is provided, particularly for improving ovulation in female animals, promoting estrus, promoting estrus synchronization, and enhancing pregnancy capacity. In some specific embodiments, the pharmaceutical combination of the invention is used to improve the reproductive performance of female animals, particularly for improving estrus mating rate, estrus conception rate, parturition rate, utilization rate, total litter size, and total healthy piglets. Therefore, the invention also relates to a method of improving the reproductive performance of female animals using the above-mentioned pharmaceutical combination, comprising administering the first substance and the second substance to the female animal during the estrous cycle. Such a method may be for non-therapeutic purposes and belongs to livestock production methods that modify the growth characteristics of animals through non-surgical treatment. In some other embodiments, the invention also includes a method for promoting estrus synchronization in female animals. Furthermore, the invention relates to the use of the pharmaceutical combination in the preparation of kits, reagent kits, or pharmaceuticals for the above-mentioned purposes and methods. Since the first substance and the second substance of the invention do not interact when in the same formulation, the first substance and the second substance can be prepared in separate containers or in the same container.

[0061] In another aspect of the invention, the use of a first substance and a second substance, and a composition comprising the first substance and the second substance, in the treatment and prevention of reproductive diseases in female animals, and in the preparation of a medicament or medicament for the treatment and prevention of reproductive diseases in female animals are provided.

[0062] In another aspect of the invention, a method of using a kit or capsule comprising a first substance and a second substance is provided. Since the first and second substances of the invention do not interact when in the same formulation, they can be injected simultaneously or separately. In another embodiment, long-acting FSH at different concentrations (e.g., 30-40 ug / ml) and hCG at different concentrations (e.g., 200-500 IU / ml) can be mixed and injected together.

[0063] The applicant was surprised to find that the drug combination provided by this invention achieves particularly advantageous improvements in enhancing the reproductive performance of female livestock, including significantly improving estrus and conception rates in sows and other female animals by combining FSH-type follicle-stimulating hormone drugs with LH-type drugs, thereby significantly improving reproductive efficiency. This will be demonstrated below through specific examples and experimental data. It should be noted that the following examples and experimental cases are merely illustrative and do not imply that the technical solution of this invention can only be implemented through these specific instances. Attached Figure Description

[0064] Figure 1. Ultrasound image of the ovary of sow No. 6 in Experiment A of Example 3.

[0065] Figure 2. Ultrasound image of the ovary of sow No. 3 in Experiment C of Example 3.

[0066] Figure 3. Ultrasound image of ovarian cyst in sow No. 6 of Experiment D in Example 3. Detailed Implementation

[0067] Example 1: Preparation method of long-acting FSH and hCG formulation

[0068] The first substance of this invention, long-acting FSH, can be an FSH-Fc fusion protein or an FSH-CTP fusion protein, for example, see application documents CN107540748A (sequence 6 or sequence 8) and CN108676096A (sequence 3 or sequence 5), respectively. In the following embodiments, unless otherwise specified, the FSH-Fc fusion protein (whose structure includes sequences 1 and 8) is mainly selected as a representative long-acting FSH. In addition, the inventors have also verified the use of other fusion proteins (including FSH-CTP fusion proteins and other FSH-Fc fusion proteins, etc.) as long-acting FSH in some embodiments. However, this does not mean that only the fusion proteins in the embodiments can achieve this; on the contrary, all FSH molecules that can play a long-acting role should be considered within the scope of protection of this application.

[0069] The second substance used in conjunction with long-acting FSH can be luteinizing hormone (LH), or long-acting luteinizing hormone (such as LH-CTP, CN115991792A), or human chorionic gonadotropin (hCG, CN108264549A), or long-acting human chorionic gonadotropin (such as hCG-Fc, CN109336981A). In the following examples, recombinant hCG (synthesized by Beijing Weijiexin Biotechnology Co., Ltd.) is selected as the second substance. In some embodiments, an ovulation-inducing drug is further used to induce ovulation in female animals. Such an ovulation-inducing drug can be hCG or its analogues (such as goserelin, gonadorelin, etc.). In the following examples, recombinant hCG (synthesized by Beijing Weijiexin Biotechnology Co., Ltd.) is selected as the ovulation-inducing drug.

[0070] After screening, the same formulation solution was found to be suitable for both long-acting FSH and hCG. The formulation solution consists of 10-50 mMPB, 0.1-1% mannitol, 1-3% sucrose, and 0.001-0.05% polysorbate 80. Validation showed that formulating 10-2000 μg / ml of long-acting FSH, 100-200000 IU / ml of hCG, or simultaneously formulating 90000 U / ml of long-acting FSH and 200000 IU / ml of hCG into this formulation solution maintained stable quality. Therefore, this formulation solution is suitable for both long-acting FSH and hCG. Furthermore, the efficacy is not affected whether long-acting FSH and hCG are prepared as a mixture for female animal reproduction, or prepared separately and injected simultaneously or sequentially.

[0071] Example 2: Effect of combined use of long-acting FSH and hCG on improving reproductive performance of gilts

[0072] 150 healthy three-way crossbred gilts weighing 130-150kg and free from reproductive diseases were selected and divided into a blank control group, a long-acting FSH monotherapy group, an hCG monotherapy group, a long-acting FSH+hCG group, and a (long-acting FSH+hCG) / hCG group, with 30 gilts in each group.

[0073] On any day of the estrus cycle (Day 0), long-acting FSH and / or hCG were administered intramuscularly to the neck of sows according to the dosage and timing specified in Table 1. Sows in the control group received an equal volume of physiological saline (2 ml / sow). Three days after administration (Day 3), sows in the (long-acting FSH+hCG) / hCG group received an additional 200 IU of hCG intramuscularly, while sows in other groups received an equal volume of physiological saline (2 ml / sow). Estrus was induced by boars every morning and afternoon after administration. Estrus status was recorded within 7 days of administration of long-acting FSH. Artificial insemination was performed on sows in estrus. Conception was assessed 25-28 days after insemination, and farrowing was recorded around day 114. The estrus-mating rate, estrus-conception rate, farrowing rate, utilization rate, total number of piglets, and total number of healthy piglets were recorded for different groups (Table 2).

[0074] In Table 1, the usage of the "long-acting FSH+hCG group" or "(long-acting FSH+hCG) / hCG group" involves sequential injections with minimal time difference in this embodiment. The second agent is injected immediately after the first agent is injected at the same location. The inventors have determined through experiments that injecting long-acting FSH first, followed by hCG, or vice versa, does not affect the experimental results. Furthermore, the inventors have also determined through experiments that pre-preparing long-acting FSH and hCG into a combined composition does not affect the experimental results either.

[0075] Table 1. Dosage table for sows in different groups

[0076] Table 2 Reproductive performance data of sows in different groups

[0077] The results of estrus and conception in different groups of sows were analyzed. Regarding the induction of estrus in sows, compared with the blank control (16.7%), neither injection of long-acting FSH (long-acting FSH monotherapy group, 20.0%) nor hCG (hCG monotherapy group, 23.3%) significantly increased the estrus and mating rate. However, injection of both long-acting FSH and hCG on day 0 (long-acting FSH+hCG group) significantly increased the estrus and mating rate (86.7%), far exceeding the sum of the effects of the two monotherapy groups, thus achieving a beneficial synergistic improvement. Since the main physiological function of hCG is generally considered to be ovulation promotion, the effect of this long-acting FSH+hCG group in increasing the estrus and mating rate was unexpected. Furthermore, administering a second injection of hCG after simultaneous injection of long-acting FSH and hCG (in the (long-acting FSH+hCG) / hCG group) further improved the conception rate during estrus in sows (88.0% vs 69.2%). The above administration did not significantly affect the farrowing rate, but the combined administration on Day 0 significantly improved the overall utilization rate of sows. Moreover, the combined administration significantly increased the total number of piglets born and the total number of healthy piglets, approximately five times higher than the control group and the single-drug group.

[0078] Therefore, compared with no medication or administration of long-acting FSH or hCG alone, combined injection of long-acting FSH and hCG into sows can improve estrus rate. Continuing hCG injection after combined administration can improve conception rate during estrus, thereby increasing sow utilization and litter size. It is also worth noting that when using wild-type FSH, due to its short half-life, even when used in combination with hCG on day 0, it only achieves a similar effect to the hCG monotherapy group. The inventors also found that in the long-acting FSH monotherapy group, because its estrus and conception effects are weaker than the dual-drug group, the estrus and mating rate is poor. Even continuing hCG administration on day 3 to promote ovulation cannot achieve the same overall significant improvement as the long-acting FSH+hCG group.

[0079] Furthermore, based on the same experimental design, the inventors further verified the use of other similar fusion proteins (including FSH-hCTP fusion protein and FSH-hFc fusion protein based on sequences 3 and 6) as long-acting FSH. The results showed that, consistent with the experimental results of the aforementioned fusion proteins, compared with no drug administration and administration of long-acting FSH or hCG alone, combined injection of long-acting FSH fusion protein and hCG into sows significantly improved the estrus and mating rate (reaching over 80%), and overall improved the sow's reproductive utilization rate, litter size, and number of healthy piglets.

[0080] Example 3: Effects of different doses of long-acting FSH and hCG combination drugs on improving reproductive performance of gilts

[0081] One hundred and eighty healthy, post-prime, and reproductively disease-free three-way crossbred gilts weighing 130-150 kg were randomly divided into six groups: a blank control group and experimental groups A, B, C, D, and E, with 30 gilts in each group. The long-acting FSH was administered intramuscularly to the neck of the gilts on any day during estrus (Day 0) and three days after administration (Day 3), according to the dosages in Table 3. The blank control group gilts received an equal volume of physiological saline (2 ml / gilt). Estrus was induced by boars in the morning and afternoon daily after administration, and the estrus status of the gilts was recorded for seven days after administration of the long-acting FSH. Artificial insemination was performed on estrus-prone gilts. Conception was assessed in gilts 25-28 days after insemination, and farrowing was recorded around day 114. Seven days after administration, abdominal ultrasound was used to monitor the ovarian status of the gilts and observe for the presence of ovarian cysts. Record the estrus mating rate, estrus conception rate, farrowing rate, utilization rate, total number of piglets born, and total number of healthy piglets of sows in different groups (Table 4).

[0082] Table 3. Dosage Table for Sows in Different Groups

[0083] Table 4 Reproductive performance data of sows in different groups

[0084] The estrus, conception, farrowing, and piglet production outcomes of sows in different groups were analyzed. The estrus mating rate, estrus conception rate, farrowing rate, and piglet utilization rate of sows in experimental groups A / B / C / D / E were significantly higher than those in the blank control group. Among them, the estrus conception rate of sows in experimental groups C and D was slightly lower. Abdominal ultrasound monitoring of the sows' ovarian status revealed large, non-ovulating follicles in some sows in both groups (Figure 1), indicating that excessive long-acting FSH and hCG may overstimulate the sow's ovaries.

[0085] The above results indicate that injecting sows with 30-50 μg of long-acting porcine FSH and 200-400 IU of hCG, followed by continued injection of 200-400 IU of hCG, can improve the estrus and conception rates of sows, thereby increasing the utilization rate of sows and the total number of piglets born.

[0086] Example 4: Batch production of long-acting FSH and hCG combined for estrus synchronization and timed insemination in gilts and multiparous sows.

[0087] One hundred healthy, post-pubertal, reproductively healthy gilts (i.e., sows that have never farrowed) aged 7.5 months were randomly divided into a blank control group and a treatment group, with 50 sows in each group. On any day of the estrous cycle, all sows were fed acetaminophen at 5 ml (20 mg) / sow / day at 2 PM for 18 consecutive days. Forty-two hours after stopping the medication, the corresponding drug was injected intramuscularly into the neck of each sow (2 ml / sow) according to the group and dosage listed in Table 5. Eighty hours after the injection, 400 IU hCG was injected intramuscularly. Artificial insemination was performed 24 hours after the injection, followed by a second insemination 8-16 hours later. Pregnancy was checked 25-28 days after insemination, and sows farrowed 114-116 days later.

[0088] One hundred healthy, multiparous sows with 5-9 litters and no reproductive diseases were selected and randomly divided into a multiparous blank control group and a multiparous drug-treated group, with 50 sows in each group. 24 hours after weaning, the corresponding drugs were administered intramuscularly to the neck of each sow (2 ml / sow) according to the group and dosage listed in Table 6. 72 hours later, 200 IU of hCG was injected intramuscularly into the neck. Artificial insemination was performed 24 hours after the injection, followed by a second insemination 8-16 hours later. Pregnancy was checked 25-28 days after insemination, and sows farrowed 114-116 days later.

[0089] Record the estrus mating rate, estrus conception rate, farrowing rate, utilization rate, average number of piglets born, average number of healthy piglets, average number of stillbirths, and average number of mummified litters of sows in different groups.

[0090] Table 5. Dosing schedule for different groups of gilts

[0091] Table 6. Dosage Table for Different Groups of Multiparous Sows

[0092] Table 7. Data on estrus, conception, and farrowing in sows of different groups.

[0093] Table 8. Farrowing data of sows in different groups

[0094] The study analyzed the estrus, conception, farrowing, and piglet distribution outcomes of gilts and multiparous sows. Compared to the control group, administration of the drug to gilts and multiparous sows increased estrus and conception rates by 8 and 16 percentage points, respectively; increased estrus conception rates by 2.7 and 6.2 percentage points, respectively; increased overall utilization rates by 10 and 24 percentage points, respectively; and increased total litter size by 88 and 163 piglets, respectively. This indicates that the combined use of long-acting FSH and hCG can be used in timed insemination programs for gilts and multiparous sows, improving reproductive efficiency, extending the productive lifespan of multiparous sows, and increasing economic benefits for pig farms.

Claims

1. A pharmaceutical combination for improving the reproductive performance of female livestock, comprising a first substance and a second substance, wherein: The first substance is a long-acting FSH fusion protein; as well as The second substance is an LH hormone.

2. The drug combination of claim 1, wherein the long-acting FSH fusion protein comprises an FSH moiety and a long-acting fragment, and the FSH moiety and the long-acting fragment are linked by a linker or not, wherein the long-acting fragment is selected from the Fc fragment and the CTP fragment of the β subunit of human chorionic gonadotropin.

3. The pharmaceutical combination of claim 1, wherein the LH hormone comprises luteinizing hormone (LH), long-acting luteinizing hormone, or human chorionic gonadotropin (hCG) and its analogues or functional equivalents.

4. The drug combination of claim 1, wherein the long-acting FSH fusion protein is an FSH-Fc fusion protein or an FSH-CTP fusion protein, and wherein the LH hormone is hCG.

5. A medicine box for improving the reproductive performance of female livestock, comprising the drug combination according to any one of claims 1 to 4, and excipients.

6. The medicine box of claim 5, wherein the first substance and the second substance are present in the medicine box in a separately packaged form.

7. A method for improving the reproductive performance of female livestock, the method comprising: On any day of the female animal's estrous cycle, the combination of drugs according to any one of claims 1 to 4 shall be administered to the female animal.

8. The method of claim 7, further comprising: Three days after the administration of the drug combination, the female animal was given a second substance.

9. The method of claim 7 or 8, further comprising: Prior to the administration of the drug combination, endoprogesterone was administered to multiple of the female animals for a period of time, thereby inducing synchronous estrus in the female animals after the administration of the drug combination.

10. Use of the pharmaceutical combination of any one of claims 1 to 4 in the preparation of a medicament box, the medicament box being used to improve ovulation in female animals, promote estrus, promote estrus synchronization, enhance fertility, or for diseases related to the reproductive system of female animals.

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