Functional low-protein daily ration for down producing goats

Abstract

The invention relates to the technical field of animal nutrition and feed, and discloses a functional low-protein daily ration for down producing goats, which is composed of a basic daily ration with crude protein content of 9.0-10.0% and an additive formed by mixing rumen bypass L-glutamine particles and L-citrulline. Aiming at the problem of reproduction rate reduction caused by low-protein daily ration confirmed for the first time, the intestinal tract is repaired through the L-glutamine, and the L-glutamine and the L-glutamine can be metabolized and converted into arginine in vivo, so that the nitric oxide level and related reproductive hormone secretion are improved. The daily ration effectively solves the problems of low nitrogen utilization rate and reduced reproductive capacity of the down producing goats under the low-protein miscellaneous meal feeding condition, remarkably improves the conception rate and the nitrogen deposition level, and has the effects of reducing the cost and reducing emission.

Description

Technical Field

[0001] This invention relates to the field of animal nutrition and feed technology, and in particular to a functional low-protein diet for cashmere goats. Background Technology

[0002] my country faces a relative shortage of protein feed resources, with significant price fluctuations leading to persistently high costs for ruminant farming. To address this challenge, reducing dietary crude protein levels and replacing expensive soybean meal with miscellaneous meals such as cottonseed meal and DDGS (corn distillers' grains and solubles) has become a primary means of cost control in intensive cashmere goat farming. However, this low-protein miscellaneous meal diet model has significant limitations in practical application. On the one hand, reduced protein intake and the presence of anti-nutritional factors in miscellaneous meals can easily lead to atrophy of intestinal villi and increased crypt depth in cashmere goats, thereby damaging the intestinal physical barrier function and reducing the body's ability to digest and absorb nitrogen and other nutrients. On the other hand, the decline in nutritional levels directly inhibits the function of the reproductive axis, resulting in abnormal estrus and ovulation in ewes and a decreased conception rate, severely restricting farming efficiency.

[0003] Studies have shown that arginine and its metabolite nitric oxide (NO) play a crucial role in regulating placental angiogenesis, improving uterine blood perfusion, and promoting embryo implantation in ruminants. However, under low-protein feeding conditions, cashmere goats exhibit a significant deficiency due to insufficient arginine synthesis substrates. Currently, the conventional approach to address this issue is to directly add arginine or its rumen-protected formulation to the feed. However, the unique physiological and metabolic mechanisms of ruminants limit the effectiveness of this method. First, unprotected free amino acids are easily degraded into ammonia by rumen microorganisms, leading to decreased nitrogen utilization. Second, even with rumen-protected technology, arginine absorbed into the portal vein is significantly decomposed by highly active arginase in the liver (i.e., the first-pass effect), resulting in extremely limited levels of arginine and nitric oxide that actually enter systemic circulation and exert reproductive regulatory effects.

[0004] Furthermore, existing nutritional regulation strategies often fall short of expectations, focusing either solely on maintaining nitrogen balance or solely on supplementing single amino acids. They lack comprehensive nutritional solutions that, in the context of low-protein diets, simultaneously address intestinal morphology repair, mitigate liver metabolic burden, and synergistically enhance reproductive performance. Therefore, how to ensure nitrogen utilization and reproductive performance in cashmere goats through metabolic restructuring of key nutrients while reducing feed protein content and costs is a pressing technical challenge for the industry. Summary of the Invention

[0005] The technical problem solved by this invention is that in the cashmere goat farming industry, the low-protein diet of mixed meal type, which is commonly used to reduce feeding costs, not only leads to a decrease in nitrogen utilization, but this invention also confirms that this feeding mode leads to a significant decline in the reproductive performance of cashmere goats. Existing single amino acid supplementation strategies are difficult to meet the multiple needs of intestinal repair, nitrogen deposition and reproductive regulation at low protein levels. Furthermore, L-glutamine is extremely unstable in the rumen and is easily degraded by microorganisms, while L-citrulline, although relatively stable, is expensive to add alone and its absorption efficiency is limited by intestinal health.

[0006] To address the above problems, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides a functional low-protein diet for cashmere goats, which consists of a low-protein basal diet and a rumen-protected compound additive. The crude protein content of the low-protein basal diet is controlled at 9.0% to 10.0% of dry matter mass. The rumen-protected compound additive is a mixture of rumen-protected L-glutamine granules and L-citrulline.

[0008] By employing the above technical solution, this invention does not simply rely on increasing protein levels, but rather achieves targeted nutritional metabolic restructuring of the "gut-kidney-reproductive axis" through specific compound additives. Its core mechanism lies in establishing a dual arginine (Arg) generation and reproductive regulation pathway: On the one hand, coating technology is used to protect L-glutamine from rumen release. As the main energy source for intestinal mucosal epithelial cells, L-glutamine directly repairs intestinal mucosal damage caused by low-protein and mixed meal diets, ensuring intestinal absorption function. At the same time, L-glutamine in small intestinal epithelial cells can be converted into endogenous citrulline.

[0009] On the other hand, exogenously added free L-citrulline is efficiently absorbed through the repaired intestines. Since citrulline is not taken up by the liver, the two streams of citrulline—endogenous conversion and exogenous absorption—converge in the blood, successfully circumventing the first-pass effect of the liver.

[0010] Both L-glutamine and L-citrulline ultimately serve as precursors to arginine, which is synthesized in large quantities in the kidneys and vascular endothelial cells via enzymatic reactions, leading to the generation of nitric oxide (NO). Endogenously generated NO acts as a vasodilator, improving blood perfusion to the ovaries and uterus and corpus luteum function, thereby promoting the secretion of gonadotropin-releasing hormone (GnRH) and progesterone, thus significantly increasing the conception rate of cashmere goats under low-protein nutritional conditions.

[0011] Preferably, in the rumen-protected compound additive, the mass ratio of L-glutamine (based on the effective component in the rumen-protected L-glutamine granules) to L-citrulline is 1 to 1.5 to 1 to 1.5.

[0012] By employing the above-mentioned technical solution, this specific ratio achieves an optimal balance between "intestinal repair" and "metabolic flux." An appropriate amount of L-glutamine prioritizes the integrity of the intestinal barrier, thus creating conditions for the efficient absorption of L-citrulline. The synergistic effect of both components significantly improves arginine production efficiency compared to a single component.

[0013] Preferably, the core material of the rumen-protected L-glutamine granules also includes vitamin B6. Specifically, by weight, the ratio of the active ingredients is: 5.0 parts L-glutamine, 5.0 parts L-citrulline, and 0.02 parts pyridoxine hydrochloride.

[0014] By adopting the above technical solution, considering that the conversion of glutamine to citrulline / arginine involves multiple deamination and transamination reactions, vitamin B6 is encapsulated inside the particles to prevent rumen degradation. After being released in the small intestine, it acts as a coenzyme for key transaminases in the form of pyridoxal-5'-phosphate, catalyzing the metabolic efficiency of the active ingredients.

[0015] Preferably, the rumen-protected L-glutamine particles have a core-shell structure and comprise the following components by weight percentage: 60% to 70% core material and 30% to 40% coating wall material; the coating wall material is composed of hydrogenated vegetable oil and stearic acid, and the mass ratio of hydrogenated vegetable oil to stearic acid is 3:1.

[0016] By adopting the above technical solution, a pH-sensitive release system for easily degradable L-glutamine was constructed using the dense lattice structure formed by hydrogenated vegetable oil and stearic acid in a specific ratio, ensuring that it accurately reaches the small intestine in its original form.

[0017] Preferably, the low-protein basal diet is a mixed meal type diet, the raw materials of which include, by weight percentage: 65% to 75% roughage, 10% to 20% energy feed, and 8% to 12% protein feed; wherein, the protein feed is selected from one or a combination of cottonseed meal, corn distillers grains and their solubles.

[0018] By adopting the above technical solution, this invention has demonstrated that even when using unconventional protein raw materials such as cottonseed meal and DDGS containing anti-nutritional factors, and reducing the protein level to 9.0%–10.0%, the production and reproductive performance of conventional soybean meal-based diets can still be maintained at a level no lower than that of conventional soybean meal-based diets through the metabolic regulation of the above-mentioned compound additives, thereby significantly reducing feeding costs.

[0019] Secondly, this invention provides a method for preparing a functional low-protein diet for cashmere goats. This method mainly includes three key steps: preparation of a rumen-protected compound additive, formulation of a low-protein basal diet, and final mixing and feeding. Specifically, firstly, rumen-protected L-glutamine granules are prepared, and then mixed with L-citrulline to obtain a compound additive; subsequently, a basal diet is formulated according to a predetermined low-protein nutritional level; finally, the rumen-protected compound additive is added to the concentrate supplement of the basal diet and mixed evenly. The daily dosage of the rumen-protected compound additive is controlled at 11.0g to 14.0g per goat.

[0020] By employing the above-mentioned technical solution, the core of this preparation method lies in step-by-step implementation and precise mixing. Considering that the proportion of rumen-protected additives in the diet is extremely low, directly mixing them into the total mixed ration (TMR) can easily lead to uneven distribution, thereby affecting the stability of individual intake. Therefore, a step-by-step dilution and mixing strategy of "additive-concentrate-basal diet" is adopted to ensure that each cashmere goat can ingest sufficient active ingredients, thereby ensuring the uniformity of feeding effects at the group level.

[0021] Preferably, the preparation process of the rumen-protected composite additive adopts a fluidized bed coating process, specifically as follows: the core material is placed in a fluidized bed, and the inlet air temperature is controlled at 42°C to 50°C to keep it in a fluidized state; then, the molten coating wall material is uniformly sprayed onto the surface of the core material at a rate of 12 mL / min to 15 mL / min and an atomization pressure of 0.25 MPa to 0.28 MPa; after the spraying is completed, the fluidization and cooling process is continued and the material is sieved.

[0022] By adopting the above technical solution, the process parameters are set based on a comprehensive consideration of the melting point characteristics of the wall material and the film-forming quality. The inlet air temperature is controlled within the range of 42℃ to 50℃, ensuring good spreadability of the molten wall material upon contact with the core material, while preventing dripping of the wall material or thermal degradation of the core components due to excessive temperature. Combined with specific atomization pressure and spray rate, the wall material droplets form a dense, micron-sized superimposed layer on the core surface. This layered physical structure effectively eliminates micropores, significantly improving the moisture resistance and abrasion resistance of the particles, ensuring the product maintains structural integrity during feed processing and rumen peristalsis.

[0023] Thirdly, the present invention provides an application of the above-mentioned functional low-protein diet for cashmere goats in improving the reproductive performance or nitrogen utilization rate of cashmere goats under low-protein feeding conditions. The diet of the present invention is applied to improve the reproductive performance or nitrogen utilization rate of cashmere goats under low-protein feeding conditions.

[0024] By adopting the above technical solution, this invention breaks through the traditional concept that high-protein diets are necessary to maintain high production performance. Regarding nitrogen utilization, through the Gln-Cit binary metabolic regulation provided by the diet, the nitrogen recycling mechanism in the body is activated. This allows cashmere goats to significantly improve their apparent nitrogen digestibility and deposited nitrogen levels even when consuming diets containing cottonseed meal or DDGS and other miscellaneous meals with a protein level of only 9.0%–10.0%, effectively reducing nitrogen emissions and achieving the ecological farming goal of "low nitrogen input, high nitrogen output".

[0025] Preferably, the improvement in reproductive performance is specifically manifested in increasing the conception rate of cashmere goats during their first estrus period and promoting the secretion of gonadotropin-releasing hormone (GnRH) and progesterone (P4).

[0026] By adopting the above-mentioned technical solution, the improvement in reproductive performance directly benefits from the dual regulation of the vascular and endocrine systems by the diet. The nitric oxide produced by metabolism improves the microcirculation of the reproductive organs, increasing the nutrient supply to follicles and the corpus luteum. Physiological monitoring data shows that this diet can significantly increase the basal levels of serum GnRH and progesterone. This optimized endocrine environment promotes the dominant development of follicles and embryo implantation, resulting in a significant increase in the first estrus conception rate in production practice, thus solving the industry's pain point of low fertility in ewes under low nutritional levels.

[0027] In summary, the present invention has at least one of the following beneficial technical effects: 1. This invention systematically studies and confirms the negative impact of low-protein diets on the reproductive rate of cashmere goats and proposes targeted nutritional regulation solutions. A binary synergistic system of "rumen-crossing L-glutamine + free L-citrulline" was constructed. L-glutamine acts as an intestinal energy repair barrier and provides an endogenous precursor, while L-citrulline, as an exogenous precursor, is directly absorbed into the bloodstream. Both ultimately converge on the arginine synthesis pathway. This mechanism not only improves blood perfusion of the reproductive system through nitric oxide-mediated vasodilation, but also significantly increases GnRH and progesterone levels, thereby reversing the decline in reproductive performance under low-protein conditions and achieving a first-estrus conception rate of over 70%.

[0028] 2. This invention defines a specific mass ratio of L-glutamine to L-citrulline, which aligns with the saturation kinetics of intestinal transporters, effectively avoiding absorption competition caused by excessive amounts of a single component. Furthermore, the introduction of vitamin B6 as a coenzyme for a key transaminase catalyzes the metabolic flow of glutamine to citrulline, further amplifying the biological efficacy of the active ingredient in vivo, enabling significant physiological regulatory effects even with low-dose addition.

[0029] 3. This invention utilizes the dense lattice structure formed by hydrogenated vegetable oil and stearic acid in a specific ratio to prepare a composite additive with excellent rumen-to-peritoneal stability. This coating layer effectively shields against rumen microbial degradation, while rapidly disintegrating under the acidic environment of the abomasum and the action of small intestinal lipases. This pH- and enzyme-sensitive release characteristic ensures that functional amino acids can precisely reach the small intestinal absorption sites in their original form, significantly improving the product's bioavailability.

[0030] 4. This invention demonstrates that even when crude protein levels are reduced to 9.0%–10.0% and cottonseed meal, DDGS, and other miscellaneous meals are used to replace expensive soybean meal, the production performance of cashmere goats can reach or even exceed that of conventional high-protein diets by adding the compound additive of this invention. This technical effect provides a feasible low-protein, low-cost diet formulation solution for the livestock industry, helping to alleviate the pressure of protein raw material shortages while reducing nitrogen emissions, resulting in significant economic and ecological benefits. Attached Figure Description

[0031] Figure 1 This is a comparison chart of the effects of different dietary treatments on nitrogen digestibility and deposited nitrogen in cashmere goats according to the present invention; wherein, (a) is a comparison chart of apparent nitrogen digestibility, and (b) is a comparison chart of deposited nitrogen. Figure 2 The diagram shows the effects of different amino acid combinations of the present invention on plasma metabolites and antioxidant function of cashmere goats; wherein, (a) is a comparison of plasma arginine and citrulline concentrations, (b) is a comparison of plasma nitric oxide (NO) levels, (c) is a comparison of plasma ornithine concentrations, and (d) is a comparison of total antioxidant capacity (T-AOC). Figure 3 The diagram shows the effects of different dietary treatments of the present invention on the reproductive performance and key hormones of cashmere goats; (a) is a comparison of conception rates during the first estrus period, (b) is a comparison of gonadotropin-releasing hormone (GnRH) levels, and (c) is a comparison of progesterone levels. Detailed Implementation

[0032] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only for illustrating the present invention and are not intended to limit the scope of the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the present invention shall fall within the scope of the present invention.

[0033] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0034] L-Glutamine (CAS No.: 56-85-9, purity ≥99.0%), L-citrulline (CAS No.: 372-75-8, purity ≥98.5%), pyridoxine hydrochloride (vitamin B6, CAS No.: 58-56-0, purity ≥98%), and DL-methionine (CAS No.: 59-51-8, purity ≥99%) are all commercially available feed-grade or food-grade additives. Hydrogenated vegetable oil (CAS No.: 68334-28-1, melting point 58℃~62℃), stearic acid (CAS No.: 57-11-4), defatted rice bran, and zeolite powder are all commercially available qualified excipients. Cottonseed meal (crude protein ≥40%), corn distillers grains and solubles (DDGS, crude protein ≥26%), corn, wheat bran, sheepgrass (crude protein ≥8.5%), and ruminant-specific compound premix are all commercially available products that meet national feed hygiene standards.

[0035] Preparation Example 1: This preparation example provides a rumen-protected compound additive (a mixture of L-glutamine-coated particles and L-citrulline), the preparation steps of which are as follows: (1) Preparation of rumen-protected L-glutamine granules: Weigh 5.0 kg of L-glutamine as the core material and place it in a fluidized bed coating machine. Separately weigh 1.61 kg of hydrogenated vegetable oil and 0.53 kg of stearic acid (mass ratio 3:1), mix and melt them to use as the coating wall material liquid. Control the inlet air temperature of the fluidized bed to 45℃ to 50℃, the atomization pressure to 0.25 MPa, and the spraying rate to 15 mL / min. Spray the wall material liquid evenly onto the surface of L-glutamine, cool and sieve to obtain rumen-protected L-glutamine granules with a coating rate of 30%.

[0036] (2) Mixing: Place the rumen-protected L-glutamine granules obtained above and 5.0 kg of L-citrulline in a mixer and mix for 10 minutes until uniform.

[0037] In this product, the mass ratio of L-glutamine (in coated form) to L-citrulline (in free form) is 1:1.

[0038] Preparation Example 2: This preparation example provides a rumen-protected compound additive, the preparation steps of which are as follows: (1) Preparation of rumen-protected L-glutamine granules: 6.0 kg of L-glutamine was weighed as the core material. Based on a 30% coating rate, 1.93 kg of hydrogenated vegetable oil and 0.64 kg of stearic acid were weighed as wall materials. The same fluidized bed process as in Preparation Example 1 was used for coating, with the inlet air temperature controlled at 48℃ and the atomization pressure at 0.28 MPa, to obtain rumen-protected L-glutamine granules.

[0039] (2) Mixing: Mix the above particles with 4.0 kg of L-citrulline until uniform, and the mixture is obtained.

[0040] In this product, the mass ratio of L-glutamine to L-citrulline is 1.5:1.

[0041] Preparation Example 3: This preparation example provides a rumen-protected compound additive, the preparation steps of which are as follows: (1) Preparation of rumen-protected L-glutamine granules: 4.0 kg of L-glutamine was weighed as the core material. Based on a 30% coating rate, 1.29 kg of hydrogenated vegetable oil and 0.43 kg of stearic acid were weighed as wall materials. The same fluidized bed process as in Preparation Example 1 was used for coating, with the inlet air temperature controlled at 46℃ and the atomization pressure at 0.26 MPa, to obtain rumen-protected L-glutamine granules.

[0042] (2) Mixing: Mix the above particles with 6.0 kg of L-citrulline until uniform, and the mixture is obtained.

[0043] In this product, the mass ratio of L-glutamine to L-citrulline is 1:1.5.

[0044] Preparation Example 4: This preparation example provides a rumen-enhanced compound additive containing vitamin B6, and its preparation steps are as follows: (1) Preparation of rumen-protected L-glutamine granules containing B6: Weigh 5.0 kg of L-glutamine and 0.02 kg of pyridoxine hydrochloride, premix them evenly and use them as the core material (Note: Vitamin B6 needs to be coated to prevent rumen degradation). Weigh 1.61 kg of hydrogenated vegetable oil and 0.54 kg of stearic acid as the wall material. Use the same process as in Preparation Example 1 for coating, and adjust the spraying rate to 12 mL / min to ensure dense coating.

[0045] (2) Mixing: Mix the above coated particles with 5.0 kg of L-citrulline until uniform, and the mixture is obtained.

[0046] In this product, vitamin B6 and L-glutamine are protected by the rumen, while L-citrulline is added directly.

[0047] Preparation Example 5: This preparation example provides a rumen-protected composite additive with high coating efficiency, and its preparation steps are as follows: (1) Preparation of L-glutamine particles with high coating rate: 5.0 kg of L-glutamine was weighed as the core material. To achieve a 40% coating rate, 2.5 kg of hydrogenated vegetable oil and 0.83 kg of stearic acid were weighed as wall materials. Using the same process as in Preparation Example 1, the spraying time was extended and the air inlet temperature was controlled at 42°C to 45°C to obtain rumen-passed L-glutamine particles with a coating rate of 40%.

[0048] (2) Mixing: Mix the above particles with 5.0 kg of L-citrulline until uniform, and the mixture is obtained.

[0049] Preparation Example 6: This preparation example provides a single-component L-citrulline additive for subsequent comparative verification.

[0050] Preparation method: Weigh 10.0 kg of L-citrulline directly to obtain the product.

[0051] Preparation Example 7: This preparation example provides a single-component rumen-exposed L-glutamine additive for subsequent comparative verification.

[0052] Preparation method: 10.0 kg of L-glutamine was weighed as the core material; 3.21 kg of hydrogenated vegetable oil and 1.07 kg of stearic acid were weighed as the coating wall material; the coating was carried out using the same process parameters as in Preparation Example 1. The core component of the obtained product was a single L-glutamine, with a coating rate of 30%.

[0053] Example 1: Low-protein basal diet of mixed meal type (Diet A) This embodiment provides a low-protein total mixed diet (TMR) with cottonseed meal and DDGS as the main protein ingredients, serving as the base diet for the example and some comparative examples. This diet aims to simulate the unconventional protein ingredient diet structure currently used in the livestock industry to reduce costs, with its crude protein level controlled at around 9.6%.

[0054] The raw material composition (air-dried basis) of this diet is shown in Table 1, and the nutrient levels (dry matter basis) are shown in Table 2.

[0055] Table 1. Raw material composition of low-protein basal diets consisting of mixed oilseed meals:

[0056] Note: The premix is ​​a commercially available compound premix for mutton sheep, containing per kilogram: copper 300-625mg; iron 1000-10000mg; zinc 2000-5000mg; manganese 2000-5000mg; cobalt 10-50mg; selenium 10-20mg; iodine 20-100mg; vitamin A 200-500KIU; vitamin D3 100-200KIU; vitamin E ≥1000IU.

[0057] Table 2. Nutritional levels of low-protein basal diets consisting of mixed oilseed meals:

[0058] Note: Metabolizable energy is a calculated value, while the other indicators are measured values.

[0059] Example 2 of Diet Formulation: Soybean Meal-Based Low-Protein Basal Diet (Diet B) This embodiment provides a low-protein total mixed diet with soybean meal as the sole protein source, serving as a basal diet for positive control. This diet maintains an isonitrogenous and isoenergetic design similar to diet formulation example 1, aiming to eliminate the interference of total energy and protein on the experimental results and to examine the influence of protein source alone.

[0060] The raw material composition (air-dried basis) of this diet is shown in Table 3, and the nutrient level (dry matter basis) is shown in Table 4.

[0061] Table 3. Raw material composition of soybean meal-based low-protein basal diets:

[0062] Note: The premix is ​​the same as Table 1.

[0063] Table 4. Nutritional levels of soybean meal-based low-protein basal diets:

[0064] Note: Metabolizable energy is a calculated value, while the other indicators are measured values.

[0065] Example 1: Functional low-protein diet for cashmere goats The diet described in this embodiment consists of the low-protein mixed meal basal diet described in Diet Formulation Example 1 and the rumen-protected compound additive obtained in Preparation Example 1.

[0066] Specific application method: Based on the diet formulation example 1, add the compound additive obtained in preparation example 1 at a dose of 12.1g per sheep per day (this dose provides 5.0g of active ingredients L-glutamine and 5.0g of L-citrulline), and mix the additive into the concentrate supplement and feed it.

[0067] Example 2: Functional low-protein diet for cashmere goats containing vitamin B6 The diet described in this embodiment consists of the low-protein mixed meal basal diet described in Diet Formulation Example 1 and the rumen-enhanced compound additive containing vitamin B6 obtained in Preparation Example 4.

[0068] Specific application method: Based on the diet formulation example 1, add the compound additive obtained in preparation example 4 at a dose of 12.2g per sheep per day (this dose provides active ingredients L-glutamine 5.0g, L-citrulline 5.0g, and pyridoxine hydrochloride 0.02g), and mix the additive into the concentrate supplement and feed it.

[0069] Example 3: Functional low-protein diet for cashmere goats The diet described in this embodiment consists of the low-protein base diet of mixed meal type described in diet formulation embodiment 1 and the compound additive obtained in preparation embodiment 2.

[0070] Specific application method: Based on the diet formulation example 1, add the compound additive obtained in preparation example 2 (this dose provides 6.0g of active ingredients L-glutamine and 4.0g of L-citrulline) to each sheep at a dose of 12.6g per sheep per day, and feed the additive into the concentrate supplement.

[0071] Example 4: Functional low-protein diet for cashmere goats The diet described in this embodiment consists of the low-protein base diet of mixed meal type described in diet formulation example 1 and the compound additive obtained in preparation example 3.

[0072] Specific application method: Based on the diet formulation example 1, add the compound additive obtained in preparation example 3 (this dose provides 4.0g of active ingredients L-glutamine and 6.0g of L-citrulline) to each sheep at a dose of 11.7g per sheep per day, and feed the additive into the concentrate supplement.

[0073] Example 5: Functional low-protein diet for cashmere goats The diet described in this embodiment consists of the low-protein basal diet of mixed meal type described in Diet Formulation Example 1 and the compound additive obtained in Preparation Example 5.

[0074] Specific application method: Based on the diet formulation example 1, add the compound additive obtained in preparation example 5 (this dose provides 5.0g of active ingredients L-glutamine and 5.0g of L-citrulline) to each sheep at a dose of 13.3g per sheep per day, and mix the additive into the concentrate supplement and feed it.

[0075] Comparative Example 1: Low-protein diets consisting of mixed oilseed meals The diet described in this comparative example uses the low-protein mixed meal basal diet described in Example 1 of the diet formulation, and no rumen-exposed amino acid additives are added during the feeding process.

[0076] Comparative Example 2: Soybean Meal-Based Low-Protein Diets The diet described in this comparative example uses the soybean meal-based low-protein basal diet described in Example 2 of the diet formulation, and no rumen-exposed amino acid additives are added during the feeding process.

[0077] Comparative Example 3: Single Citrulline Low-Protein Diet The diet described in this comparative example consists of the mixed meal type low-protein basal diet described in Diet Formulation Example 1 and the single L-citrulline additive obtained in Preparation Example 6.

[0078] Specific application method: Based on the diet formulation example 1, add the additive obtained in preparation example 6 at a dose of 10.0g per sheep per day (since citrulline does not need to be coated, this dose is equivalent to 10.0g of the active ingredient L-citrulline), and mix the additive into the concentrate supplement and feed it.

[0079] Comparative Example 4: Low-protein diet with mono-glutamine The diet described in this comparative example consists of the mixed meal type low-protein basal diet described in Diet Formulation Example 1 and the single L-glutamine additive obtained in Preparation Example 7.

[0080] Specific application method: Based on the diet formulation example 1, add the additive obtained in preparation example 7 (equivalent to 10.0g of active ingredient L-glutamine) at a dose of 14.3g per sheep per day, and mix the additive into the concentrate supplement and feed it.

[0081] Comparative Example 5: Non-rumen-protected low-protein diet The diet described in this comparative example consists of a low-protein basal diet of mixed meal type as described in Diet Formulation Example 1 and uncoated amino acid ingredients.

[0082] Specific application method: Based on the diet formulation example 1, add raw material grade L-glutamine at a dose of 5.0g per sheep per day and raw material grade L-citrulline at a dose of 5.0g per sheep per day, and feed the raw materials directly into the concentrate supplement.

[0083] Test Example 1: Nitrogen Metabolism and Apparent Nutrient Digestibility Determination The experimental steps are as follows: (1) Twenty-four adult non-pregnant cashmere goats in good condition and with similar weight (approximately 35.5 kg ± 1.2 kg) were randomly divided into four groups of six each. The experiment adopted a single-factor randomized grouping design, with each group corresponding to Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 5, respectively. All experimental goats were housed in independent metabolic cages to allow for separate collection of feces and urine.

[0084] (2) The experiment lasted for 15 days, with the first 10 days being the pre-trial period, during which sheep adapted to the metabolic cage environment and the experimental diet; the last 5 days were the formal trial period, during which all feces and urine were collected. During the experiment, sheep were fed twice a day at 08:00 and 16:00, with free access to water. The additives in Example 1 and Comparative Example 5 were fed together with the morning concentrate, and it was ensured that the sheep had finished eating the roughage before feeding.

[0085] (3) During the trial period, the feed intake and uneaten feed of each group of sheep were accurately recorded daily. Feces were collected using the total feces collection method. After weighing the collected feces daily, 10% tartaric acid nitrogen fixation was added at 10% of the total weight, mixed well, and stored at -20℃. At the same time, urine was collected using the total urine collection method. 100mL of 10% sulfuric acid was added to the urine collection bucket in advance to prevent ammonia volatilization. The daily urine volume was recorded, and samples were taken and mixed for storage.

[0086] (4) After the experiment, the frozen fecal and urine samples were thawed and mixed evenly. The dry matter (DM), organic matter (OM) and total nitrogen (N) contents in feed, feed residues and fecal samples were determined according to the AOAC (2000) standard method. The total nitrogen content in urine samples was determined, and the apparent digestibility of nutrients, deposited nitrogen and nitrogen utilization rate were calculated accordingly.

[0087] The experimental data are shown in Table 5.

[0088] Table 5. Results of apparent nutrient digestibility and nitrogen metabolism indicators in each group of cashmere goats:

[0089] Table 5 Data and Figure 1 This invention reveals the reshaping effect of the present invention on nitrogen metabolism efficiency in the context of a low-protein mixed meal diet. In Example 1, when fed with low-quality protein sources such as cottonseed meal and DDGS, the apparent nitrogen digestibility of cashmere goats reached 66.77%, and the deposited nitrogen increased to 6.92 g / d. This result is not only significantly better than Comparative Example 1 (mixed meal control group, with a nitrogen digestibility of only 54.76%), but more importantly, its value has statistically matched or even slightly exceeded that of Comparative Example 2 (high-quality soybean meal group). This indicates that by supplementing with rumen-treated glutamine and citrulline, the negative impact of anti-nutritional factors in mixed meals on intestinal absorption function is eliminated, the intestinal mucosal barrier is repaired, and the bioavailability of inexpensive protein sources reaches the standard of expensive protein sources, achieving efficient conversion of low-quality raw materials.

[0090] The difference between Example 1 and Comparative Example 5 confirms the necessity of rumen protection for key components. With the addition of glutamine and citrulline, the nitrogen utilization rate of Comparative Example 5 (uncoated) (33.82%) was significantly lower than that of Example 1 (44.13%). This is mainly because uncoated L-glutamine is extremely unstable in the rumen and is easily and rapidly degraded into ammonia by microorganisms, thus losing its function as a repair agent for small intestinal epithelial cells. When the intestinal barrier is not effectively repaired, the body's overall absorption capacity of nutrients is limited. This also means that even though L-citrulline is relatively stable in the rumen, its subsequent absorption and metabolic conversion efficiency is greatly reduced due to poor intestinal function. Only by ensuring that glutamine can reach the small intestine in its original form to exert its energy-supplying function through the technical solution of this invention can the efficient absorption of dietary nitrogen throughout the intestine be promoted.

[0091] In terms of nitrogen deposition data, Example 1 showed a 91% improvement compared to Comparative Example 1, further demonstrating that this invention does not simply provide a nitrogen source, but rather reduces urinary nitrogen excretion (from 4.88 g / d to 3.55 g / d) by optimizing the in vivo amino acid profile, thus retaining more nitrogen in the body for tissue synthesis and reproductive system reserves. This dual mechanism of improved digestion and reduced excretion lays the material foundation for subsequent improvements in reproductive performance.

[0092] Test Example 2: Verification of Plasma Amino Acid Metabolism and Antioxidant Mechanism The experimental steps are as follows: (1) The experimental subjects were grouped in the same way as in Test Example 1. On the 5th day of the trial period (i.e. the last day of the entire feeding cycle), before morning feeding, blood was collected from the jugular vein of all the experimental sheep in Example 1 (compound group), Example 2 (B6-added group), Comparative Example 3 (monocitrulline group) and Comparative Example 4 (monoglutamine group).

[0093] (2) Collect 10 mL of blood using a vacuum blood collection tube containing heparin sodium anticoagulant. Immediately after collection, invert the tube to mix and prevent coagulation. Store the blood in an ice box and transport it back to the laboratory. Centrifuge the sample at 3000 r / min for 15 minutes at 4℃, aspirate the supernatant plasma, dispense it into 1.5 mL centrifuge tubes, and store them in an ultra-low temperature freezer at -80℃ for later testing.

[0094] (3) After the plasma samples were thawed, they were treated with sulfosalicylic acid to remove protein, and the concentration of free amino acids (citrulline, arginine, ornithine) in the plasma was determined using a fully automated amino acid analyzer.

[0095] (4) A separate sample of plasma was collected, and the nitric oxide (NO) content was determined using the nitrate reductase method, the superoxide dismutase (SOD) activity was determined using the hydroxylamine method, and the glutathione peroxidase (GSH-Px) activity and total antioxidant capacity (T-AOC) were determined using a colorimetric method. All biochemical indicators were measured strictly in accordance with the kit instructions, and each sample was measured three times and the average value was taken.

[0096] The experimental data are shown in Table 6.

[0097] Table 6. Results of plasma amino acid metabolism and antioxidant indicators in cashmere goats of each group:

[0098] Table 6 data and Figure 2This study reveals the metabolic regulatory mechanism of the "gut-kidney-reproductive axis" in this invention. The plasma arginine level (138.56 μmol / L) and NO level (0.046 μmol / L) in Example 1 were both higher than those in Comparative Example 3 (monocitrulline group), even though the amount of citrulline added in Comparative Example 3 was actually twice that of Example 1. This non-linear synergistic effect confirms the key role of glutamine, which not only repairs intestinal epithelial cells and enhances the absorption efficiency of exogenous citrulline, but also converts itself into endogenous citrulline within the small intestinal epithelial cells, forming a dual-source synergistic effect. In contrast, although Comparative Example 4 (monoglutamine group) had decent antioxidant capacity, its ability to increase plasma arginine (95.34 μmol / L) was far less than that of the compound group due to limitations in enzyme conversion rate, proving that glutamine alone cannot overcome metabolic bottlenecks.

[0099] The data from Example 2 highlight the synergistic value of vitamin B6 as a metabolic coenzyme; after the introduction of vitamin B6, the plasma ornithine concentration significantly increased to 89.12 μmol / L, significantly higher than in Example 1. Ornithine is a key intermediate in the urea cycle, and its increased level indicates that the activity of ornithine transaminase (OAT) is effectively supported by a cofactor, thereby accelerating substrate flux. This accelerated metabolic flux further increased the concentration of the final product NO (up to 0.051 μmol / L), providing a more abundant blood perfusion signal for the reproductive system.

[0100] Regarding the antioxidant mechanism, Examples 1 and 2 both demonstrated superior stress resistance compared to single-component formulations. Glutamine, as a precursor to glutathione (GSH), directly enhanced GSH-Px activity. Furthermore, NO generated from citrulline is itself a signaling molecule that can induce the expression of antioxidant enzymes. The combined use of these two formulations achieved a synergistic antioxidant protection effect, with the significant increase in T-AOC levels indicating a better redox balance, which is crucial for maintaining high-intensity metabolic activity during the mating season and protecting oocytes from oxidative damage.

[0101] Test Example 3: Verification of Reproductive Performance and Endocrine Regulation The experimental steps are as follows: (1) Experimental subjects and grouping: The experimental subjects were grouped in the same way as in Test Example 1; covering Examples 1 to 5 and Comparative Examples 1 and 3. All ewes selected for the experiment were multiparous nonpregnant ewes, and B-ultrasound examination was performed before the start of the experiment to confirm that there were no reproductive system diseases.

[0102] (2) Synchronization of estrus: To accurately calculate the conception rate during the first estrus period, the "CIDR (vaginal suppository) + PGF2α" method was used for estrus synchronization. A CIDR suppository (containing 300 mg of progesterone) was inserted on day 12 of the trial period, and the suppository was removed on day 24, followed by an intramuscular injection of 0.1 mg of cloprostenol (PGF2α).

[0103] (3) Blood hormone assay: Blood was collected from the jugular vein 48 hours after thrombectomy (peak estrus) and plasma was separated. The concentrations of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and progesterone (P4) were determined by radioimmunoassay (RIA).

[0104] (4) Mating and Confirmation of Conception: Within 48 to 72 hours after thrombectomy, estrus is detected by using a ram to test for estrus, and artificial insemination is performed on ewes in estrus. 35 days after mating, the gestational sac and fetal heartbeat are examined by ultrasound to confirm pregnancy, and the conception rate of the first estrus is calculated accordingly.

[0105] The experimental data are shown in Table 7.

[0106] Table 7. Statistics on reproductive hormone levels and conception rates in each group of cashmere goats:

[0107] Note: Conception rate data are based on the statistical average of multiple repeated trials.

[0108] Table 7 and Figure 3 The data presents the final reproductive output benefits of the technical solution of this invention. Compared with Comparative Example 1, which was fed a diet of ordinary mixed oilseed meals, Example 1 achieved a significant increase in conception rate (from 40.0% to 72.5%). The significant recovery of GnRH and P4 levels indicates that the additive relieved the inhibition of the hypothalamic-pituitary-gonadal axis (HPG axis) caused by nutritional deficiency, restoring the normal reproductive rhythm of ewes. This result confirms that even under low-protein diet conditions of only 9.6%, ideal reproductive returns can be achieved through precise amino acid nutrition regulation.

[0109] Comparing the data of Example 1 (compound group) and Comparative Example 3 (pure citrulline group), it can be seen that although the addition of high dose of citrulline alone can also improve reproductive performance to a certain extent (conception rate 65.0%), Example 1 achieved a better effect by constructing a dual mechanism of glutamine repair of the intestine and citrulline bypass metabolism (conception rate 72.5%).

[0110] From a hormonal perspective, the levels of GnRH (289.45 ng / L) and progesterone P4 (4.82 ng / mL) in Example 1 were significantly higher than those in Comparative Example 3 (265.8 ng / L and 4.23 ng / mL, respectively). This indicates that while nitric oxide production from citrulline alone can improve blood flow, the overall metabolic state of the body is not optimal without the underlying repair and energy support of glutamine for intestinal absorption. The compounding strategy of this invention not only utilizes citrulline to directly synthesize arginine but also utilizes glutamine to ensure efficient absorption of nutrients and provide endogenous precursors. This strategy of simultaneously increasing precursors and repairing the intestines achieves a biological effect greater than the sum of its parts (1+1>2).

[0111] Furthermore, in Example 2, the conception rate further increased to 75.0% after the introduction of vitamin B6, and the GnRH level reached the highest level in the entire group at 305.12 ng / L. This verifies the catalytic role of coenzyme factors in enhancing the reproductive metabolic flux of amino acids, providing a better technical option for breeding scenarios pursuing ultimate reproductive performance. In summary, this invention established the optimal cost-effectiveness model through Example 1 and confirmed the irreplaceable role of this nutritional regulation scheme in improving the utilization value of miscellaneous meal diets and ensuring the reproductive health of ewes.

[0112] Based on the test results from the above three dimensions, this invention constructs a systematic nutritional regulation scheme for low-protein diets consisting of mixed meal feeds. Firstly, at the digestion and absorption level, rumen-treated glutamine repairs the intestinal mucosa, increasing the nitrogen digestibility of the mixed meal feed to a level comparable to soybean meal feeds, thus overcoming the bottleneck of low raw material utilization. Secondly, at the metabolic mechanism level, through the synergistic conversion of glutamine and citrulline in the "gut-kidney axis," combined with the coenzyme effect of vitamin B6, plasma arginine and NO levels are significantly increased, enhancing the body's antioxidant defense capabilities. Finally, in production applications, this metabolic improvement successfully translates into a breakthrough in reproductive performance, significantly increasing the conception rate of cashmere goats under low-protein diet conditions while maintaining hormone secretion homeostasis. This technical system not only achieves efficient utilization of non-grain protein feeds and reduces feeding costs, but also resolves the contradiction between nutrition and reproduction in ruminants through endogenous physiological regulation mechanisms, demonstrating significant economic benefits and promotional value.

[0113] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A functional low-protein diet for cashmere goats, characterized in that, The diet includes a low-protein basal diet and a rumen-protected compound additive; The crude protein content of the low-protein basal diet is 9.0% to 10.0% of the dry matter mass. The rumen-protected compound additive is composed of rumen-protected L-glutamine particles and L-citrulline; The diet delivers L-glutamine through rumen-exposed L-glutamine granules, which then releases L-glutamine into the intestines. This, in conjunction with free L-citrulline, increases plasma arginine and nitric oxide levels, repairs the intestinal barrier, and thus improves nitrogen metabolism and reproductive performance in cashmere goats.

2. The functional low-protein diet for cashmere goats according to claim 1, characterized in that, In the rumen-protected compound additive, based on the effective components in the rumen-protected L-glutamine granules, the mass ratio of L-glutamine to L-citrulline is (1-1.5):(1-1.5).

3. The functional low-protein diet for cashmere goats according to claim 1, characterized in that, The rumen-protected compound additive also includes vitamin B6; The vitamin B6 is contained inside the rumen-protected L-glutamine granules, and together with L-glutamine, it serves as a core material. The active ingredients are formulated in the following proportions by weight: 5.0 parts L-glutamine, 5.0 parts L-citrulline, and 0.02 parts pyridoxine hydrochloride.

4. The functional low-protein diet for cashmere goats according to claim 1, characterized in that, The rumen-passed L-glutamine particles have a core-shell structure and comprise the following components by weight percentage: Core materials: 60%–70%; Wall covering material: 30%–40%; The core material is L-glutamine or a mixture of L-glutamine and vitamin B6; the coating wall material is composed of hydrogenated vegetable oil and stearic acid, and the mass ratio of hydrogenated vegetable oil to stearic acid is 3:

1.

5. The functional low-protein diet for cashmere goats according to claim 1, characterized in that, The rumen-protected compound additive is prepared using the following process: (1) Preparation of rumen-passed L-glutamine particles: L-glutamine is placed in a fluidized bed as the core material, and the inlet air temperature is controlled at 42℃~50℃ to make it fluidized; the molten coating wall material is sprayed onto the surface of the core material at a rate of 12mL / min~15mL / min and an atomization pressure of 0.25MPa~0.28MPa; after the spraying is completed, the fluidized cooling is performed and the particles are sieved. (2) Mixing: Mix the rumen-protected L-glutamine granules obtained in step (1) with L-citrulline in a certain proportion to obtain the final product.

6. The functional low-protein diet for cashmere goats according to claim 1, characterized in that, The low-protein basal diet is a mixed meal diet, and its ingredients, by weight percentage, include: Roughage: 65%–75%; Energy feed: 10%–20%; Protein feed: 8%–12%; The protein feed is selected from one or a combination of cottonseed meal, corn distillers grains and their solubles.

7. A method for preparing a functional low-protein diet for cashmere goats as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of the rumen-protected compound additive; (2) Formulate the low-protein basal diet as described in claim 1; (3) Add the rumen-protected compound additive obtained in step (1) to the concentrate supplement of the low-protein basal diet described in step (2) and mix evenly; The amount of the rumen-protected compound additive added is 11.0g to 14.0g per sheep per day.

8. The application of the functional low-protein diet for cashmere goats as described in any one of claims 1-6 in improving the reproductive performance or nitrogen utilization of cashmere goats under low-protein feeding conditions.

9. The application according to claim 8, characterized in that, The improvement in reproductive performance is specifically manifested in increasing the conception rate of cashmere goats during their first estrus period and promoting the secretion of gonadotropin-releasing hormone and progesterone.

10. The application according to claim 8, characterized in that, The improvement in nitrogen utilization is specifically manifested in increasing the apparent nitrogen digestibility and deposited nitrogen level of cashmere goats under the condition of feeding diets containing cottonseed meal or corn distillers grains and their solubles.

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