A biological composition for delaying reproductive function decline and its preparation method and application

Abstract

The application discloses a biological composition for delaying reproductive function decline, which comprises liposome-coated nano selenium, L-carnitine or a salt thereof, vitamin E or a derivative thereof, L-arginine, beta-carotene and an organic metal salt. Compared with the prior art, the application is based on multiple mechanisms of oxidative damage, energy metabolism disorder, microcirculation disorder and insufficient hormone synthesis involved in reproductive function decline, and multiple components are synergized, so that the composite reproductive function decline is effectively delayed, the reproductive life is significantly prolonged, and the comprehensive effect is better than that of the prior art.

Description

Technical Field

[0001] This invention belongs to the field of avian biomedicine, specifically relating to a biological composition for delaying the decline of reproductive function, its preparation method, and its application. Background Technology

[0002] The decline of male reproductive function is a complex physiological process involving multiple factors and pathways. Under high metabolic load conditions, the reproductive system faces multiple challenges, including increased oxidative damage, energy metabolism disorders, and microcirculatory disturbances. These three factors are mutually causal, forming a vicious cycle of "increased metabolic load → oxidative damage → deterioration of the reproductive microenvironment → functional decline." How to effectively slow down this decline process and extend the reproductive lifespan of males has been a long-standing and important issue in the field of reproductive biology, but current intervention strategies still face many bottlenecks.

[0003] First, existing nutritional regulation programs mostly employ simple combinations of single or two active ingredients, lacking synergistic design based on biochemical pathways among the components. This makes it difficult to simultaneously address multiple issues such as oxidative stress, energy depletion, and impaired microcirculation, resulting in limited effectiveness in delaying complex reproductive function decline. Second, intervention programs generally adopt a uniform supply model throughout the entire life cycle, failing to consider the significant differences in nutritional requirements of the reproductive system at different developmental stages (tissue differentiation, spermatogenesis initiation, and function maintenance), leading to a mismatch between supply and actual demand. Third, the blood-testis barrier, as one of the tightest blood-tissue barriers in the body, significantly restricts the targeted delivery of exogenous active ingredients to testicular tissue. Conventional formulations of vitamin E and selenium have unsatisfactory transport efficiency and target tissue enrichment, making it difficult to achieve effective concentrations in the spermatogenic microenvironment. Fourth, existing evaluation methods mainly rely on conventional semen parameter testing, which is time-consuming and cannot sensitively reflect changes in the oxidative stress state of the reproductive microenvironment, thus failing to support dynamic adjustments to intervention programs. Summary of the Invention

[0004] To address at least some of the technical problems existing in the prior art, the present invention provides a biological composition and its formulation, preparation method and application, which achieves the goal of delaying the decline of male reproductive function by multi-target synergistic design and nano-targeted delivery across the blood-testis barrier.

[0005] In a first aspect, the present invention provides a biological composition for delaying the decline of reproductive function, comprising liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivative, L-arginine, β-carotene and organometallic salts.

[0006] In some embodiments, the organometallic salt includes organozinc and / or calcium salts.

[0007] In some embodiments, the liposome-encapsulated selenium nanoparticles comprise a bilayer shell composed of electronegative lipids and a selenium nanoparticle core.

[0008] In some embodiments, the lipid bilayer is composed of lecithin and sterol, and the mass ratio of lecithin to sterol is 2-10:1.

[0009] In some embodiments, the encapsulation efficiency of the liposome-coated selenium nanoparticles is ≥89%.

[0010] In some embodiments, the average particle size of the liposome-coated selenium nanoparticles is 20-100 nm.

[0011] In some embodiments, the vitamin E or its derivative is selected from one or more of tocopherol, tocotrienol, and dl-α-tocopherol acetate.

[0012] In some embodiments, the L-carnitine or its salt is selected from one or more of L-carnitine, L-carnitine tartrate, and L-carnitine hydrochloride.

[0013] In some embodiments, the organic zinc salt is selected from one or more of zinc glycinate, zinc lactate, zinc citrate, and zinc methionine.

[0014] In some embodiments, the biological composition includes: liposome-coated selenium nanoparticles, L-carnitine, natural vitamin E, L-arginine, β-carotene, and zinc methionine.

[0015] In some embodiments, the biological composition comprises, by weight, 0.1-2 parts of liposome-coated nano-selenium, 20-80 parts of L-carnitine or its salt, 50-150 parts of vitamin E or its derivative, 20-60 parts of L-arginine, 10-30 parts of β-carotene, and 5-15 parts of organometallic salt.

[0016] In some embodiments, the biological composition further includes an unsaturated hydroxy fatty acid selected from at least one of 5-hydroxy-2-hexenoic acid lactone, 7-hydroxy-2-octenoate ethyl ester, 10-hydroxy-2-decenoic acid, 3-hydroxy-5-dodecenoic acid, and 15-hydroxypentadecanenoic acid.

[0017] In some embodiments, the biological composition comprises, by weight, 0.5-5.0 parts of unsaturated hydroxy fatty acids, 0.1-2.0 parts of liposome-coated nano-selenium, 20-80 parts of L-carnitine or its salt, 50-150 parts of vitamin E or its derivative, 20-60 parts of L-arginine, 10-30 parts of β-carotene, and 5-15 parts of organometallic salt zinc.

[0018] In some embodiments, the composition further includes an acceptable carrier selected from at least one of starch, corn starch, dextrin, maltodextrin, lactose, microcrystalline cellulose, and defatted rice bran.

[0019] In a second aspect, the present invention provides a formulation comprising the aforementioned biological composition.

[0020] In some embodiments, the formulation is an oral formulation.

[0021] In some embodiments, the dosage form of the oral preparation is a powder, granules, solution, or suspension.

[0022] Thirdly, the present invention provides a method for preparing the aforementioned biological composition, which includes weighing and mixing liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivative, L-arginine, β-carotene, and organometallic salts in a specified ratio to obtain the composition.

[0023] In some embodiments, the preparation method includes in-situ reduction of a selenium source in a liposome dispersion system to generate selenium nanoparticles, while simultaneously achieving liposome encapsulation of the selenium nanoparticles.

[0024] In some embodiments, the preparation method includes adding a selenium source and a reducing agent to a liposome dispersion system to carry out a reduction reaction, so that the selenium nanoparticles are generated in situ and simultaneously coated in a phospholipid bilayer.

[0025] Fourthly, the present invention provides the use of a biological composition in the preparation of a product for delaying reproductive function decline, said biological composition comprising liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivatives, L-arginine, β-carotene and organometallic salts.

[0026] In some implementations, the delay in reproductive function decline includes at least one of the following: increasing semen volume, increasing sperm density, increasing sperm motility, reducing sperm abnormality rate, increasing effective sperm count, increasing fertilization rate of hatching eggs, increasing hatching rate of hatching eggs, increasing chick survival rate, or extending breeding life.

[0027] Fifthly, the present invention provides a method for improving the reproductive performance of male birds, comprising applying the aforementioned biological composition or the aforementioned preparation to male birds.

[0028] In some embodiments, the biological composition or formulation is administered at differentiated doses at different life stages of male birds.

[0029] In some implementations, the different life stages include the testicular Sertoli cell proliferation window, the testicular development and spermatogenesis initiation window, and the high-frequency sperm collection and reproductive maintenance window.

[0030] Compared with the prior art, the present invention has the following beneficial effects: 1. Synergistic effect of multiple targets to systematically delay complex reproductive dysfunction. This invention overcomes the limitations of simply combining single or two active ingredients. Based on the multiple mechanisms involved in reproductive function decline, including oxidative damage, energy metabolism disorders, microcirculatory disturbances, and insufficient hormone synthesis, it designs a multi-component synergistic solution covering the entire chain of "antioxidant network remodeling—energy metabolism support—microcirculation improvement—hormone synthesis promotion." Each active ingredient works synergistically through clearly defined biochemical pathways to achieve immediate antioxidant defense and enzyme activity repair, energy supply and circulatory support, and hormone synthesis promotion, systematically solving the problem of multi-factor complex decline.

[0031] 2. Precise supply based on phased biological events to match differentiated needs across different life cycles. Based on the key biological events in the development and maintenance of the male reproductive system, this invention designs differentiated supply programs for each life stage, so that the types and dosages of nutritional support are precisely matched with the actual needs of different window periods (tissue differentiation period, spermatogenesis initiation period, and function maintenance period), avoiding insufficient efficiency or waste of resources caused by the mismatch between supply and actual needs under the "one-size-fits-all" model.

[0032] 3. Liposome nanoparticle-targeted delivery crosses the blood-testis barrier, overcoming the key bottleneck of tissue enrichment of active ingredients. This invention employs a phospholipid bilayer-coated selenium nanoparticle delivery system. By leveraging the fusion or endocytosis of liposomes with the cell membrane, it crosses the blood-testis barrier to deliver the active ingredient directly to testicular tissue. Compared to ordinary selenium nanoparticles, liposome encapsulation significantly increases the selenium accumulation in the target tissue, demonstrating the targeting advantage of this delivery system in crossing the blood-testis barrier. This is the first time that highly efficient enrichment of selenium nanoparticles in male reproductive target tissues has been achieved.

[0033] 4. A rapid evaluation and dynamic adjustment mechanism to achieve closed-loop management of intervention programs. This invention establishes a rapid multi-index evaluation method based on oxidative stress and antioxidant capacity, which can reflect the redox state of the reproductive microenvironment in a timely manner. Based on this feedback, the intervention plan can be dynamically adjusted, realizing a closed-loop management of "evaluation → feedback → adjustment → re-evaluation". This overcomes the shortcomings of existing technologies where fixed plans are difficult to adapt to dynamic changes in individual states.

[0034] 5. Significant overall effects, effectively extending reproductive lifespan. System verification shows that this invention can significantly improve multiple reproductive function indicators, effectively prolong the reproductive lifespan of male individuals, and has outstanding comprehensive effects and practical application value in delaying the decline of reproductive function. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] the term In this article, the term "active ingredient" refers to substances that can produce physiological effects that delay the decline of reproductive function, including liposome-coated selenium nanoparticles, vitamin E and its derivatives, L-carnitine and its salts, L-arginine, β-carotene, organometallic salts, and optional unsaturated hydroxy fatty acid compounds.

[0039] In this document, the term "carrier" refers to an excipient or diluent used to carry the aforementioned active ingredients and nutritional components. It does not possess significant therapeutic or nutritional activity, and its main functions are to improve the physical properties of the composition, provide appropriate volume and mass for uniform mixing in feed, and adsorb oily components to improve powder flowability. The carrier is not an active ingredient of this invention; it is only used to disperse and carry the aforementioned active substances and does not participate in producing the stated physiological effects.

[0040] The carriers applicable to this invention include, but are not limited to, one or more mixtures of the following substances: corn starch, maltodextrin, wheat bran powder, defatted rice bran, zeolite powder, calcium carbonate, and dicalcium phosphate. Corn starch or maltodextrin is preferred as the carrier because it has the following advantages: low price and wide availability; regular particle shape, good flowability, easy mixing and sieving; moderate hygroscopicity, not affecting the storage stability of the composition; and no adverse effect on animal palatability.

[0041] In this document, the term "unsaturated hydroxy fatty acid" refers to a fatty acid compound containing at least one carbon-carbon double bond (C=C) and simultaneously having one or more hydroxyl (-OH) substituents, including its free acid form and its acceptable derivative forms (such as lactones, esters, etc.). In this application, "unsaturated hydroxy fatty acid" is preferably a fatty acid having a C6-C15 straight-chain or branched alkenyl backbone and being substituted with one or more hydroxyl groups.

[0042] For example, the unsaturated hydroxy fatty acids include, but are not limited to, at least one of 5-hydroxy-2-hexenoic acid lactone (C6, containing a lactone ring), 7-hydroxy-2-octenoate ethyl ester (C8, ethyl ester form), 10-hydroxy-2-decenoic acid (C10, free acid), 3-hydroxy-5-dodecenoic acid (C12, free acid), and 15-hydroxypentadecanenoic acid (C15, free acid).

[0043] Composition In one aspect, the present invention provides a biological composition for delaying the decline of reproductive function, the active ingredients of which include liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivatives, L-arginine, β-carotene and organometallic salts.

[0044] The liposome-coated selenium nanoparticles comprise a bilayer shell composed of electronegative lipids and a selenium nanoparticle core. The electronegative lipid bilayer is composed of phospholipids and sterols, with a phospholipid to sterol mass ratio of 2-10:1, for example, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. The phospholipids are selected from one or more of soybean lecithin, egg yolk lecithin, hydrogenated phospholipids, and synthetic phospholipids (such as DPPC and DSPC); the sterols are selected from one or more of similar components with membrane-stabilizing effects, such as cholesterol, dihydrocholesterol, and phytosterols (such as β-sitosterol). The average particle size of the liposome-coated selenium nanoparticles is 20-100 nm, for example, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm.

[0045] In some embodiments, the composition, by weight, comprises: 0.1-2.0 parts of liposome-coated selenium nanoparticles, for example, 0.1, 0.4, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 parts; 20-80 parts of L-carnitine or its salt, for example, 20, 30, 40, 50, 60, 70, or 80 parts; and vitamin E or its derivative. 50-150 parts, for example 50, 60, 80, 100, 130, or 150 parts; L-arginine 20-60 parts, for example 20, 25, 30, 40, 50, or 60 parts; β-carotene 10-30 parts, for example 10, 15, 20, 25, or 30 parts; organometallic salt 5-15 parts, for example 5, 7, 10, 12, or 15 parts. In some embodiments, it also includes 100-300 parts of a carrier, for example 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 parts.

[0046] In some embodiments, the active ingredients of the biological composition, by weight, include 1.0 part of liposome-coated selenium nanoparticles, 50 parts of L-carnitine, 100 parts of natural vitamin E, 40 parts of L-arginine, 20 parts of β-carotene, and 10 parts of zinc methionine (calculated as elemental zinc), with 200 parts of corn starch as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a mass ratio of soybean lecithin to cholesterol of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0047] In some embodiments, the active ingredients, by weight, include 1.5 parts of liposome-coated selenium nanoparticles, 60 parts of L-carnitine tartrate, 120 parts of dl-α-tocopherol acetate, 50 parts of L-arginine, 20 parts of β-carotene, and 12 parts of zinc glycine (calculated as elemental zinc), with 250 parts of maltodextrin as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a mass ratio of soybean lecithin to cholesterol of 5:1. The average particle size of the liposome-coated selenium nanoparticles is 45 nm, and the encapsulation efficiency is 91.5%.

[0048] In some embodiments, the active ingredients of the composition, by weight, include 0.8 parts of liposome-coated selenium nanoparticles, 40 parts of L-carnitine, 80 parts of natural vitamin E, 30 parts of L-arginine, 15 parts of β-carotene, and 8 parts of zinc lactate (calculated as elemental zinc), with 150 parts of defatted rice bran as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a mass ratio of soybean lecithin to cholesterol of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0049] In some embodiments, the active ingredient of the composition further includes an unsaturated hydroxy fatty acid selected from at least one of 5-hydroxy-2-hexenoic acid lactone, 7-hydroxy-2-octenoate ethyl ester, 10-hydroxy-2-decenoic acid, 3-hydroxy-5-dodecenoic acid, and 15-hydroxypentadecanenoic acid.

[0050] In some embodiments, the composition comprises, by weight, 0.5-5.0 parts of unsaturated hydroxy fatty acids, for example, 0.5 parts, 0.7 parts, 0.9 parts, 1.1 parts, 1.3 parts, 1.5 parts, 1.8 parts, 2.0 parts, 2.4 parts, 2.8 parts, 3.2 parts, 3.6 parts, 4.0 parts, 4.5 parts, or 5.0 parts, preferably 1.0-2.0 parts; 0.1-2.0 parts of liposome-coated nano-selenium, for example, 0.1 parts, 0.4 parts, 0.8 parts, 1.0 parts, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts, or 2.0 parts; and 20-80 parts of L-carnitine or its salt, for example, 20 parts, 30 parts, 40 parts, 50 parts, or 60 parts. 70 parts, 80 parts; 50-150 parts of vitamin E or its derivatives, such as 50 parts, 60 parts, 80 parts, 100 parts, 130 parts, 150 parts; 20-60 parts of L-arginine, such as 20 parts, 25 parts, 30 parts, 40 parts, 50 parts, 60 parts; 10-30 parts of β-carotene, such as 10 parts, 15 parts, 20 parts, 25 parts, 30 parts; 5-15 parts of organometallic salts, such as 5 parts, 7 parts, 10 parts, 12 parts, 15 parts; and 100-300 parts of carriers, such as 100 parts, 120 parts, 140 parts, 160 parts, 180 parts, 200 parts, 220 parts, 240 parts, 260 parts, 280 parts, 300 parts.

[0051] In some embodiments, the content of unsaturated hydroxy fatty acids in the biological composition of this application is designed to be administered to poultry at an application rate of 0.01-0.15 g per day.

[0052] In some embodiments, the active ingredients of the composition, by weight, include 1.0 part of 5-hydroxy-2-hexenolide, 1.0 part of liposome-coated selenium nanoparticles, 50 parts of L-carnitine, 100 parts of natural vitamin E, 40 parts of L-arginine, 20 parts of β-carotene, and 10 parts of zinc methionine (calculated as elemental zinc), with 200 parts of corn starch as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a soybean lecithin to cholesterol mass ratio of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0053] In some embodiments, the active ingredients of the composition, by weight, include 1.0 part of 10-hydroxy-2-decenoic acid, 1.0 part of liposome-coated selenium nanoparticles, 50 parts of L-carnitine, 100 parts of natural vitamin E, 40 parts of L-arginine, 20 parts of β-carotene, and 10 parts of zinc methionine (calculated as elemental zinc), with 200 parts of corn starch as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a soybean lecithin to cholesterol mass ratio of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0054] In some embodiments, the active ingredients of the composition, by weight, include 1.0 part of ethyl 7-hydroxy-2-octenate, 1.0 part of liposome-coated selenium nanoparticles, 50 parts of L-carnitine, 100 parts of natural vitamin E, 40 parts of L-arginine, 20 parts of β-carotene, and 10 parts of zinc methionine (calculated as elemental zinc), with 200 parts of corn starch as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a soybean lecithin to cholesterol mass ratio of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0055] In some embodiments, the active ingredients of the composition, by weight, include 1.0 part of 15-hydroxypentadecanoic acid, 1.0 part of liposome-coated selenium nanoparticles, 50 parts of L-carnitine, 100 parts of natural vitamin E, 40 parts of L-arginine, 20 parts of β-carotene, and 10 parts of zinc methionine (calculated as elemental zinc), with 200 parts of corn starch as the carrier. The electronegative lipid bilayer of the liposome-coated selenium nanoparticles is composed of soybean lecithin and cholesterol, with a soybean lecithin to cholesterol mass ratio of 4:1. The average particle size of the liposome-coated selenium nanoparticles is 52 nm, and the encapsulation efficiency is 89.2%.

[0056] Preparation method In another aspect, the present invention provides a method for preparing the aforementioned biological composition, comprising weighing and mixing liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivative, L-arginine, β-carotene, and organometallic salts in a specified ratio to obtain the composition. In some embodiments, the method further includes mixing unsaturated hydroxy fatty acids with the above-mentioned components to obtain the composition.

[0057] In some embodiments, the preparation method of the aforementioned composition includes adding a selenium source and a reducing agent to a liposome dispersion system to carry out a reduction reaction, so that the nano-selenium particles are generated in situ and simultaneously coated in the phospholipid bilayer.

[0058] In some embodiments, the liposomes are composed of soybean lecithin and cholesterol, and the method for preparing the liposomes coated with selenium nanoparticles includes: (1) Soybean lecithin and cholesterol are dissolved in an organic solvent, and the organic solvent is removed by vacuum evaporation to form a lipid film; (2) Hydrate the lipid film by adding a hydration medium to obtain a blank liposome suspension; (3) Add selenium source and reducing agent to the blank liposome suspension to carry out reduction reaction and generate nano-selenium particles, wherein some nano-selenium particles are encapsulated inside the liposomes and some nano-selenium particles are attached to the surface of the liposomes. (4) The suspension obtained in step (3) is subjected to particle size homogenization treatment; (5) The homogenized suspension is dried to obtain liposome-coated selenium nanoparticles.

[0059] In some preferred embodiments, the method for preparing the liposome-coated selenium nanoparticles includes the following steps: (1) Weigh soybean lecithin and cholesterol in a mass ratio of 3:1-6:1, dissolve them in anhydrous ethanol, and evaporate them under reduced pressure in a rotary evaporator at 35-45℃ to remove the organic solvent, forming a uniform lipid film on the bottle wall. (2) Add phosphate buffer solution with pH 6.5-7.5 to the lipid membrane and hydrate it in a water bath at 40-50℃ for 30-60 minutes to obtain a blank liposome suspension; (3) Under stirring conditions, sodium selenite solution and vitamin C solution (molar ratio of sodium selenite to vitamin C is 1:4-1:6) are added to the blank liposome suspension in sequence. The reaction is carried out at room temperature in the dark for 30-60 minutes. Sodium selenite and vitamin C react in the aqueous phase to generate nano-selenium particles. Some of the nano-selenium particles are encapsulated inside the liposomes and some are attached to the surface of the liposomes. (4) The reaction suspension was extruded through polycarbonate membranes of 200 nm, 100 nm and 50 nm 5-10 times to obtain a liposome-coated nano-selenium suspension with uniform particle size. (5) Freeze-dry the suspension at -40 to -50°C for 24 to 48 hours to obtain liposome-coated nano-selenium freeze-dried powder.

[0060] In some preferred embodiments, the preparation method of the composition includes: weighing liposome-coated nano-selenium, vitamin E, L-carnitine, arginine, β-carotene, organometallic salt and carrier according to the ratio, putting them into a high-speed mixer, mixing at a speed of 200-400 rpm for 10-20 minutes, and passing them through a 60-100 mesh sieve to obtain the final product.

[0061] application Another aspect of the present invention provides the use of the composition in the preparation of a biological composition for delaying reproductive function decline, characterized in that the active ingredient of the composition comprises liposome-coated selenium nanoparticles, L-carnitine or a salt thereof, vitamin E or a derivative thereof, L-arginine, β-carotene, and organometallic salts. In some embodiments, the active ingredient of the composition further comprises unsaturated hydroxy fatty acids. The delaying of reproductive function decline includes at least one of the following: increasing semen volume, increasing sperm density, increasing sperm motility, reducing sperm abnormality rate, increasing effective sperm count, increasing fertilization rate of hatching eggs, increasing hatching rate of hatching eggs, increasing chick viability, or extending breeding life.

[0062] In another aspect, the present invention provides a method for improving the reproductive performance of male birds, comprising administering the aforementioned composition or a formulation comprising the aforementioned composition to male birds. The composition or formulation is administered at differentiated doses at different life stages of the male birds. These different life stages include the testicular Sertoli cell proliferation window, the testicular development and spermatogenesis initiation window, and the high-frequency semen collection and reproductive maintenance window.

[0063] In some implementation schemes, the following differentiated administration protocols are adopted based on the three stages of rooster spermatogenesis: the testicular Sertoli cell proliferation window, the testicular development and spermatogenesis initiation window, and the high-frequency semen collection and reproductive maintenance window. Phase 1: Early development stage (0-8 weeks old) - Testicular Sertoli cell proliferation window period The composition is added to the basal diet at a dosage ratio of 0.5%-1.0% and fed daily. The basal diet has a crude protein content of 18%-20% and a metabolizable energy of 2.8-2.90 Mcal / kg. The biological goal at this stage is to promote gonadal development and the proliferation and differentiation of testicular Sertoli cells in breeding roosters. The number of testicular Sertoli cells is fixed before sexual maturity (approximately 14 weeks of age), and their number directly determines the upper limit of spermatogenesis support capacity in adulthood (i.e., the maximum sperm production). This stage focuses on supplying nutrients that promote cell proliferation (zinc, β-carotene, arginine) to lay the "quantitative foundation" for the spermatogenesis capacity of breeding roosters throughout their lives. Excessive addition of antioxidant components (nano-selenium, vitamin E) is not recommended at this stage to avoid interfering with normal cell proliferation signaling pathways.

[0064] Phase Two: Late Development (9-20 weeks of age) - Window of Testicular Development and Spermatogenesis Initiation The composition is added to the basal diet at a dosage ratio of 1.0%-1.5% and fed daily. The basal diet has a crude protein content of 16%-17% and a metabolizable energy of 2.75-2.85 Mcal / kg. The biological goal of this stage is to promote rapid testicular tissue development and the normal initiation of spermatogenesis. The seminiferous epithelium begins to form, and spermatogonia differentiate into primary spermatocytes. This stage provides comprehensive nutrition to all target sites, promoting testicular tissue development while establishing an antioxidant defense system, thus preparing a "quality reserve" for high-frequency semen collection during the breeding season. The key to this stage is the balanced supply of "development promotion" and "antioxidant" nutrients.

[0065] Phase 3: Breeding Period (21 weeks to culling) - High-frequency semen collection and reproductive maintenance window The composition is added to the basal diet at a dosage ratio of 1.5%-2.0% and fed daily. The basal diet has a crude protein content of 15%-16% and a metabolizable energy of 2.70-2.80 Mcal / kg. The biological objective of this stage is to maintain semen quality under high-frequency semen collection conditions and delay testicular function decline. After roosters enter the high-frequency semen collection state, their testes are under high metabolic load, and oxidative stress is sharply aggravated. This stage focuses on strengthening antioxidant defense (high-dose combination of vitamin E and nano-selenium) and energy metabolism support (L-carnitine), while continuously maintaining testicular microcirculation perfusion through arginine. The key to this stage is the synergistic enhancement of "antioxidant" and "energy supply". Preferably, the addition ratio is 1.8%-2.0%.

[0066] Example This example illustrates the preparation of a composition, which includes the following steps: I. Preparation of liposome-coated selenium nanoparticles Preparation Example 1 Weigh 5 g of soybean lecithin and cholesterol at a mass ratio of 4:1, dissolve in 50 mL of anhydrous ethanol, and evaporate under reduced pressure in a rotary evaporator at 40 °C in a water bath to remove the organic solvent, forming a uniform lipid film on the bottle wall. Add 100 mL of pH 7.0 phosphate buffer to the lipid film and hydrate in a water bath at 45 °C for 45 minutes to obtain a blank liposome suspension. Under stirring, add 10 mL of 0.1 mol / L sodium selenite solution and 10 mL of 0.5 mol / L vitamin C solution (the molar ratio of sodium selenite to vitamin C is approximately 1:5) to the blank liposome suspension sequentially, and react at room temperature in the dark for 45 minutes. Sodium selenite and vitamin C react in the aqueous phase to generate selenium nanoparticles, some of which are encapsulated inside the liposomes, while others are attached to the surface of the liposomes. The resulting suspension is extruded eight times each through polycarbonate membranes of 200 nm, 100 nm, and 50 nm. The extruded suspension was freeze-dried at -45°C for 36 hours to obtain liposome-coated selenium nanoparticle lyophilized powder. Transmission electron microscopy revealed that the selenium nanoparticles had a particle size of 40-60 nm, and dynamic light scattering analysis showed an average particle size of 52 nm, with an encapsulation efficiency of 89.2%.

[0067] Preparation Example 2 Weigh 5 g of soybean lecithin and cholesterol in a 5:1 mass ratio, dissolve in 50 mL of anhydrous ethanol, and remove the organic solvent by vacuum evaporation in a rotary evaporator at 38 °C in a water bath. Add 100 mL of phosphate buffer (pH 7.4) to the lipid membrane and hydrate for 60 minutes in a 45 °C water bath. Under stirring, add 8 mL of 0.1 mol / L sodium selenite solution and 8 mL of 0.5 mol / L vitamin C solution sequentially, and react at room temperature in the dark for 60 minutes. Extrude the resulting suspension sequentially through 200 nm, 100 nm, and 50 nm polycarbonate membranes 10 times each. Freeze-dry the extruded suspension at -50 °C for 48 hours to obtain liposome-coated selenium nanoparticle lyophilized powder. The average particle size of the selenium nanoparticles was 45 nm, and the encapsulation efficiency was 91.5%.

[0068] Preparation Example 3 Soybean lecithin and cholesterol were weighed at mass ratios of 3:1 and 6:1, respectively, and the remaining procedures were the same as in Preparation Example 1. Results: The encapsulation rate of the 3:1 group was 78.3%, and the encapsulation rate of the 6:1 group was 81.5%, both lower than the encapsulation rate (above 89%) in the range of 4:1-5:1.

[0069] II. Preparation of the Composition Composition 1 Weigh the following components according to the indicated weights: 1.0 part of liposome-coated nano-selenium prepared in Example 1, 100 parts of natural vitamin E (tocopherol), 50 parts of L-carnitine, 40 parts of L-arginine, 20 parts of β-carotene, 10 parts of zinc methionine (calculated as elemental zinc), and 200 parts of corn starch. Add the above components sequentially to a high-speed mixer and mix at 300 rpm for 15 minutes. Pass the mixture through an 80-mesh sieve to obtain the final composition.

[0070] Composition 2 Weigh the following components by weight: 1.5 parts of liposome-coated nano-selenium obtained in Preparation Example 2, 120 parts of dl-α-tocopherol acetate, 60 parts of L-carnitine tartrate, 50 parts of L-arginine, 25 parts of β-carotene, 12 parts of zinc glycine (calculated as elemental zinc), and 250 parts of maltodextrin. The preparation method is the same as that of Composition 1.

[0071] Composition 3 Weigh the following components by weight: 0.8 parts of liposome-coated nano-selenium obtained in Preparation Example 1, 80 parts of natural vitamin E, 40 parts of L-carnitine, 30 parts of L-arginine, 15 parts of β-carotene, 8 parts of zinc lactate (calculated as elemental zinc), and 150 parts of defatted rice bran. The preparation method is the same as that of Composition 1.

[0072] Composition 4 Weigh the following components by weight: 5 parts 10-hydroxy-2-decenoic acid, 1.0 part liposome-coated nano-selenium obtained in Preparation Example 1, 100 parts natural vitamin E, 50 parts L-L-carnitine, 40 parts L-arginine, 20 parts β-carotene, 10 parts zinc methionine (calculated as elemental zinc), and 200 parts corn starch. The preparation method is the same as that of Composition 1.

[0073] Comparative Example To investigate the effects of different combinations of active ingredients and nano-selenium forms on delaying the decline of reproductive function, the following comparative examples were set up.

[0074] Table 1: Composition of biological compositions (parts by mass) Note: A mass portion refers to the total mass relative to 100 portions of the basal diet; the control example is feeding only the basal diet.

[0075] Among them, the ordinary nano-selenium used in Comparative Examples 1-9 and 1-10 were uncoated nano-selenium particles with an average particle size of about 50 nm, which were used to verify the promoting effect of liposome coating on crossing the blood-testis barrier and achieving targeted enrichment in testicular tissue.

[0076] Test Example 1 This embodiment is a staged application example of the composition, specifically including: 1. Select healthy roosters and feed them according to a differentiated dosage plan: Phase 1 (testicular Sertoli cell proliferation window, 0-8 weeks of age): Add 0.8% of Example Composition 1 to the basal diet (19% crude protein, 2.85 Mcal / kg), feed 3 times a day, and allow free access to water.

[0077] Second stage (testicular development and spermatogenesis initiation window, 9-20 weeks of age): Add 1.2% of Example Composition 1 to the basal diet (16.5% crude protein, 2.80 Mcal / kg), feed twice a day, with free access to water.

[0078] Phase III (High-frequency semen collection and reproductive maintenance window, 21 weeks of age to culling): Add 1.8% of the composition prepared in the examples or comparative examples to the basal diet (15.5% crude protein, 2.75 Mcal / kg), feed twice daily, with free access to water. II. Evaluation Methods Semen was collected from roosters at 45 weeks of age for quality evaluation. (1) Sperm motility and sperm density were measured using the CASA system (manufactured by Minitube, Spain); (2) The MDA content in seminal plasma was determined by the TBARS colorimetric method (the kit was purchased from Nanjing Jiancheng Bioengineering Institute). (3) The activities of T-AOC and SOD in seminal plasma were determined by ELISA (the kit was purchased from Wuhan Huamei Biotechnology Co., Ltd.); (4) The baseline values ​​were the mean values ​​of the population at 22-24 weeks of age: sperm motility 86.5%, MDA content 2.1 nmol / mL, T-AOC 11.8 U / mL, and SOD activity 82.3 U / mL.

[0079] III. Evaluation Results All data are expressed as mean ± standard deviation. One-way ANOVA was performed using SPSS 26.0 software. Differences between groups were analyzed using Duncan's new multiple range method for multiple comparisons.

[0080] Table 2: Comparison of Main Detection Indicators 1. Verification of multi-target synergistic effects 1.1 Effects of adding a single component As shown in Table 2, while the indicators of each single-component supplementation group (Comparative Example 1-1, Comparative Example 1-2, Comparative Example 1-3, and Comparative Example 1-4) were slightly better than those of the basal diet control group (control example), the improvement was limited. Taking sperm motility as an example, the best-performing group, the nano-selenium-only supplementation group, only achieved 75.8%, an improvement of only 3.5 percentage points compared to the basal diet control group (72.3%). The sperm motility of the β-carotene + organic zinc combination (two components, i.e., Comparative Example 1-5) was 73.5%, slightly better than that of a single component, but the difference from the basal diet control group was not statistically significant. This indicates that a small number of components cannot effectively solve the problem of complex reproductive decline.

[0081] 1.2 Verification of the synergistic effect of pairwise combinations In the comparative examples, the paired combinations (Comparative Examples 1-5, 1-6, 1-7, and 1-8) showed better results than the single-component groups in the comparative examples. Taking Comparative Example 1-6 (liposome-coated nano-selenium + vitamin E) as an example, its sperm motility reached 80.2%, which was significantly better than the 75.8% of the nano-selenium alone group and the 74.5% of the vitamin E alone group (P<0.05).

[0082] To rigorously verify the existence of a synergistic effect, this invention calculated the theoretical expected value of the combination of nano-selenium and vitamin E (assuming it to be the arithmetic sum of the effects of adding either ingredient individually). The specific calculation method was as follows: using the basal dietary control group as a baseline, the absolute improvement values ​​of each indicator were calculated for the nano-selenium-added group and the vitamin E-added group alone, respectively. These two values ​​were then added together to obtain the "theoretical expected value" (i.e., the effect that the combination should achieve assuming no synergistic effect). The results are shown in Table 3. Table 3: Synergistic effect analysis of nano-selenium and vitamin E combination As shown in Table 3, the actual values ​​of the antioxidant group in terms of sperm motility, MDA, T-AOC and other indicators were better than the theoretical expected values, which confirmed that there is a positive synergistic effect between nano selenium and vitamin E, rather than a simple additive effect.

[0083] 1.3 Comprehensive synergistic effect of complete five- or six-component system Comparative Examples 1-6, which showed the best results, still had significantly lower performance indicators than the complete protocols of Examples 2-1 and 2-4: sperm motility (80.2% vs 88.5%, 80.2% vs 98.5%), sperm density (3.2 vs 3.8 × 10⁻⁶). 9 / mL, 3.2 vs 4.2×10 9The differences in the concentrations of MDA (2.8 vs 2.1 nmol / mL, 2.8 vs 1.5 nmol / mL), T-AOC (10.8 vs 12.8 U / mL, 10.8 vs 15.5 U / mL), and SOD (74.5 vs 85.4 U / mL, 74.5 vs 95.1 U / mL) were all statistically significant (P<0.05).

[0084] This indicates that even the strongest two-component combination cannot replace the synergistic effect of other targets—addressing only the antioxidant problem without addressing energy metabolism (L-carnitine), microcirculation (arginine), and hormone synthesis (β-carotene + organic zinc) results in a limited overall improvement in semen quality. The five-component complete solution of this invention covers all aspects of complex reproductive decline through multi-target synergy, producing a significant synergistic effect that cannot be achieved by single components or pairwise combinations.

[0085] Furthermore, the addition of immunomodulatory 10-hydroxy-2-decenoic acid (10-hydroxy-2-decenoic acid) to the complete five-component regimen further improved all indicators compared to the five-component regimen. Specifically, the six-component regimen showed superior sperm motility, sperm volume, and sperm density compared to the five-component regimen (P<0.05), with further reductions in MDA content and increases in T-AOC and SOD activities. This indicates that 10-hydroxy-2-decenoic acid exerts its antioxidant and anti-inflammatory effects through the immunomodulatory pathway, complementing the antioxidant-energy-circulation-hormonal pathway of the original five-component regimen. Notably, the combination of 10-hydroxy-2-decenoic acid and liposome-coated selenium nanoparticles (Comparative Examples 1-8) showed weaker effects than the five-component regimen, suggesting that it requires synergy with other targets at the immunomodulatory level to achieve optimal results.

[0086] The above results confirm that the introduction of 10-hydroxy-2-decenoic acid is not a simple additive process, but rather a deep synergistic effect achieved through an independent immunomodulatory target and a multi-target network of the five components, further covering the immune imbalance involved in reproductive dysfunction. Therefore, the complete five- or six-component scheme of this invention covers all aspects of complex reproductive decline through multi-target synergy, producing a significant synergistic effect that cannot be achieved by a single component or any combination of components.

[0087] 2. Validation of the targeted delivery effect of liposome-encapsulated lipids across the blood-testis barrier In Comparative Examples 1-10, after replacing the liposome-coated selenium nanoparticles with ordinary selenium nanoparticles with the same selenium content, although the other components were exactly the same, all indicators (except MDA) of Comparative Examples 1-10 were lower than those of Composition 1 of Examples 1.

[0088] To further verify the promoting effect of liposome coating on crossing the blood-testis barrier, testicular tissue was collected from roosters in each group at the end of the experiment (60 weeks of age), and the tissue selenium content was measured. The results showed that the testicular tissue selenium content of composition 1 in Example 1 was 1.85 ± 0.21 μg / g, while that of comparative examples 1-10 was 1.06 ± 0.15 μg / g. Liposome coating increased the selenium accumulation in the testes by approximately 74.5% (P < 0.05). Correspondingly, the seminal plasma T-AOC of composition 1 in Example 1 at 45 weeks of age (12.8 U / mL) was significantly higher than that of comparative examples 1-10 (12.1 U / mL, P < 0.05).

[0089] The above results confirm that liposome encapsulation can effectively promote the crossing of the blood-testis barrier by nano-selenium and its targeted accumulation in testicular tissue, thereby significantly enhancing the actual effect of the antioxidant network in the target tissue. This invention verifies the targeted delivery effect of liposome-derived selenium nanoparticles across the blood-testis barrier in breeding roosters.

[0090] 3. Comparison of seed lifespan and fertilization rate The breeding roosters in each major experimental group were tracked and recorded until culling (culling was carried out when the natural mating fertilization rate was below 90%). The results are as follows: Table 4: Comparison of seed lifespan and reproductive performance of the main experimental groups As shown in Table 4, the breeding lifespan of roosters using the exemplary complete schemes of the present invention (compositions 1 and 4 of the embodiments) reached 22 months and 28 months, respectively, which is 10 months and 16 months longer than the basal diet control group (control example, 12 months), 4 months and 10 months longer than Comparative Examples 1-6 (18 months), and 6 months and 12 months longer than the ordinary nano-selenium complete scheme group of Comparative Examples 1-10 (16 months). All of the above differences are statistically significant (P<0.05).

[0091] Regarding the fertilization rate of hatching eggs, the fertilization rate of Example Composition 1 reached 95.6%, and that of Example Composition 4 reached 96.2%, which were 7.1 and 7.7 percentage points higher than the basal diet control group (88.5%), respectively, and 1.8 and 2.4 percentage points higher than that of Comparative Examples 1-10 (93.8%), respectively. Regarding the hatching rate, the hatching rate of Example Composition 1 was 93.8%, and that of Example Composition 4 was 94.8%, which were 8.6 and 9.6 percentage points higher than the basal diet control group (85.2%), respectively, and 2.3 and 3.3 percentage points higher than that of Comparative Examples 1-10, respectively, significantly better than each comparative example group.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biological composition for delaying the decline of reproductive function, characterized in that, These include liposome-coated selenium nanoparticles, L-carnitine or its salts, vitamin E or its derivatives, L-arginine, β-carotene, and organometallic salts.

2. The biological composition according to claim 1, characterized in that, The organometallic salts include organozinc salts and / or calcium salts.

3. The biological composition according to claim 2, characterized in that, The liposome-encapsulated selenium nanoparticles consist of a bilayer shell composed of electronegative lipids and a selenium nanoparticle core.

4. The biological composition according to claim 3, characterized in that, The bilayer is composed of phospholipids and sterols, and the mass ratio of phospholipids to sterols is 2-10:

1.

5. The biological composition according to claim 3, characterized in that, The encapsulation efficiency of the liposome-coated selenium nanoparticles is ≥89%.

6. The biological composition according to claim 3, characterized in that, The average particle size of the liposome-coated selenium nanoparticles is 20-100 nm.

7. The biological composition according to claim 1, characterized in that, The vitamin E or its derivatives are selected from one or more of tocopherol, tocotrienol, and dl-α-tocopherol acetate.

8. The biological composition according to claim 1, characterized in that, The L-carnitine or its salt is selected from one or more of L-carnitine, L-carnitine tartrate, and L-carnitine hydrochloride.

9. The biological composition according to claim 2, characterized in that, The organic zinc salt is selected from one or more of zinc glycinate, zinc lactate, zinc citrate, and zinc methionine.

10. The biological composition according to claim 2, characterized in that, include: Liposomes encapsulate nano-selenium, L-carnitine, natural vitamin E, L-arginine, β-carotene, and zinc methionine.

11. The biological composition according to claim 1, characterized in that, By weight, it includes: 0.1-2 parts of liposome-coated nano-selenium, 20-80 parts of L-carnitine or its salt, 50-150 parts of vitamin E or its derivative, 20-60 parts of L-arginine, 10-30 parts of β-carotene, and 5-15 parts of organometallic salt.

12. The biological composition according to claim 1, characterized in that, The biological composition further includes unsaturated hydroxy fatty acids selected from at least one of 5-hydroxy-2-hexenoic acid lactone, 7-hydroxy-2-octenoate ethyl ester, 10-hydroxy-2-decenoic acid, 3-hydroxy-5-dodecenoic acid, and 15-hydroxypentadecanenoic acid.

13. The biological composition according to claim 12, characterized in that, By weight, it includes 0.5-5.0 parts of unsaturated hydroxy fatty acids, 0.1-2.0 parts of liposome-coated nano-selenium, 20-80 parts of L-carnitine or its salt, 50-150 parts of vitamin E or its derivative, 20-60 parts of L-arginine, 10-30 parts of β-carotene, and 5-15 parts of organometallic salts.

14. The biological composition according to claim 1, characterized in that, It further includes an acceptable carrier selected from at least one of starch, corn starch, dextrin, maltodextrin, lactose, microcrystalline cellulose, and defatted rice bran.

15. A formulation, characterized in that, The biological composition comprising any one of claims 1-14.

16. The formulation according to claim 15, characterized in that, The preparation is an oral preparation.

17. The formulation according to claim 16, characterized in that, The dosage form of the oral preparation is powder, granules, solution or suspension.

18. The method for preparing the biological composition according to claim 1, characterized in that, The composition is obtained by coating liposomes with selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivative, L-arginine, β-carotene, and organometallic salts in a specified ratio and mixing them.

19. The preparation method according to claim 18, characterized in that, This includes the step of adding a selenium source and a reducing agent to a liposome dispersion system to carry out a reduction reaction, so that the nano-selenium particles are generated in situ and simultaneously coated in a phospholipid bilayer.

20. The use of a biological composition in the preparation of a product for delaying reproductive function decline, characterized in that, The biological composition includes liposome-coated selenium nanoparticles, L-carnitine or its salt, vitamin E or its derivatives, L-arginine, β-carotene, and organometallic salts.

21. The application according to claim 20, characterized in that, The delay in reproductive function decline includes at least one of the following: increasing semen volume, increasing sperm density, increasing sperm motility, reducing sperm abnormality rate, increasing effective sperm count, increasing fertilization rate of hatching eggs, increasing hatching rate of hatching eggs, increasing chick survival rate, or extending breeding life.

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