A supplementing concentrate for Mongolian horses in late pregnancy, a supplementing method and application

Abstract

The application provides a supplementing concentrate for Mongolian horses in the late pregnancy, a supplementing method and application, and relates to the technical field of animal nutrition and feed science.The supplementing concentrate is composed of the following raw materials in parts by weight: corn 70-72%, soybean meal 6-9%, bran 8.7-16.2%, molasses 1-5%, vegetable oil 1-3%, stone powder 0.5-1%, calcium hydrogen phosphate 0.1-1%, lysine 0.1-1%, salt 0.1-1%, and premix 0.1-1%.The application systematically explores the influence of supplementing concentrates with different energy and protein levels on the growth performance, nutrient digestion rate, blood biochemical indexes, reproductive hormone level, antioxidant performance, immune function, fecal microbial flora and metabolomics of Mongolian horses in the late pregnancy, so as to provide a theoretical basis and data support for determining a scientific supplementing scheme of energy and protein in the daily ration of Mongolian horses in the late pregnancy.

Description

Technical Field

[0001] This invention belongs to the field of animal nutrition and feed science and technology, specifically a supplementary concentrate for Mongolian horses in late pregnancy, a supplementary feeding method, and its application. Background Technology

[0002] Mongolian horses, a unique and important livestock resource in the northern grasslands of my country, possess many distinctive characteristics. From the perspective of their digestive system, horses are monogastric herbivores, a significant difference from common livestock such as pigs, cattle, and sheep. This difference permeates multiple aspects of their physiological structure, digestive process, and nutritional needs. Pigs are omnivores, and their digestive systems are adapted to diverse food sources. Their teeth are well-suited for chewing various types of feed, including plant-based ingredients and animal protein. During digestion, pigs' stomachs and small intestines secrete various digestive enzymes to effectively break down and absorb starches, proteins, and fats in concentrates. For example, pigs can digest corn and soybean meal very well, obtaining abundant energy and protein. Horses, on the other hand, primarily feed on forage, and their teeth are better suited for nibbling and grinding high-fiber pastures. Horses have relatively small stomachs and lack the powerful digestive enzyme secretion capacity of pigs to process high-concentrate diets. Overfeeding horses with concentrates, especially starchy grains, can lead to serious problems such as indigestion, colic, and even laminitis. This is because horses' digestive systems cannot process and absorb nutrients in concentrates as efficiently as pigs'. Cattle and sheep are ruminants with a unique rumination mechanism. A ruminant's stomach has four chambers: the rumen, reticulum, omasum, and abomasum. After food enters the rumen, it undergoes fermentation by microorganisms, breaking down crude fiber into volatile fatty acids and other nutrients. This fermentation process allows cattle and sheep to fully utilize the energy in roughage. In terms of nutritional supplementation, cattle and sheep can increase their protein content by adding urea. Urea can be converted into microbial protein by rumen microorganisms, providing an additional protein source for the animal. However, horses do not possess this characteristic. A horse's stomach is a single-chambered stomach, lacking rumination and the fermentation action of rumen microorganisms, making it unable to effectively convert and utilize urea. Adding urea to a horse not only fails to supplement protein but may also harm its health, such as causing ammonia poisoning. Currently, there is a lack of refined feeding standards specifically for the Mongolian horse breed. In practice, general techniques applicable to pigs, cattle, and sheep are often directly applied, leading to nutritional imbalances and low feeding efficiency. Therefore, developing precision nutrition technologies specifically adapted to the physiological and feeding characteristics of the Mongolian horse has become an urgent need to promote the development of this industry.

[0003] Meanwhile, as a local breed, the Mongolian horse has developed unique biological characteristics and adaptability through long-term natural selection and artificial breeding. It is hardy, adaptable, and disease-resistant, making it a vital pillar of local herders' livelihoods. However, in high-altitude and cold regions like Inner Mongolia, the harsh winter climate poses a significant challenge to the survival and reproduction of Mongolian horses. Long, cold winters cover natural grasslands with thick snow, causing the grass to wither and its nutritional value to drop sharply, especially crude protein and digestible energy. This coincides with the late stages of pregnancy in Mongolian horses, when the fetus grows rapidly, and the mare's demand for energy, protein, minerals, and vitamins reaches its peak. Traditional grazing methods, which rely on natural grazing, fail to adequately consider the differences in digestive and nutritional needs between horses and other animals, as well as the unique breed characteristics of the Mongolian horse, making it difficult to meet the nutritional requirements of this special stage. This leads to a negative nutritional balance in the mare, resulting in a series of production and health problems.

[0004] Currently, supplementary feeding techniques for Mongolian horses in late pregnancy during the winter and spring seasons remain relatively rudimentary. In most cases, farmers rely solely on empirical feeding methods, lacking systematic and scientific nutritional control plans. Common practices in existing techniques involve simply increasing the amount of concentrate feed or providing ordinary reserve hay. This approach fails to fully recognize the differences in digestive and nutritional needs between horses and other animals, and does not precisely tailor the feed to the physiological characteristics and nutritional requirements of mares in late pregnancy. Such methods have several significant drawbacks: First, the energy-to-protein ratio is inappropriate. Excessive energy intake can easily lead to obesity and metabolic disorders in mares, while insufficient energy intake can cause fetal stunting. This differs from the nutritional needs and responses of other animals at similar stages. For example, while pigs also require sufficient nutrition in late pregnancy, their energy tolerance range is relatively wide, and energy intake can be controlled by adjusting feed intake; horses, due to the limitations of their digestive system, require much stricter control over energy intake. Second, the quality of protein and the balance of essential amino acids are neglected, affecting the immune function of mares and fetal tissue development. Furthermore, different animals have different requirements and utilization efficiency for protein and amino acids. Cattle and sheep can synthesize some essential amino acids through microorganisms during rumination, thus having relatively lower requirements for the balance of amino acids in their feed. Horses, however, can only absorb amino acids from their feed through their small intestine, requiring higher protein quality and a more balanced intake of essential amino acids. Thirdly, the synergistic effect of minerals, vitamins, and fiber was not considered, leading to low nutrient absorption and utilization rates. This is because the horse's unique digestive system differs from other animals in its absorption and utilization mechanisms. Pigs absorb minerals and vitamins relatively directly, while horses require fiber to maintain normal intestinal peristalsis and a balanced gut microbiota to promote their absorption. Fourthly, the lack of regulation of the gut microbiota structure easily leads to digestive dysfunction. The composition and function of the horse's gut microbiota also differ from other animals. The rumen microbiota of cattle and sheep is relatively stable and highly adaptable to feed; however, the horse's gut microbiota is more susceptible to changes in feed and environmental factors, requiring more precise regulation.

[0005] Furthermore, existing supplementary feeding techniques often fail to consider the regional characteristics, breed traits, and seasonal climate variations of Mongolian horses. As a local breed, the Mongolian horse possesses unique adaptation mechanisms to the local climate and environment, but current techniques do not adequately account for these factors, leading to inconsistent supplementary feeding effects. Problems such as abortion, weak foals, delayed postpartum recovery, and prolonged breeding intervals are frequent in mares, severely hindering the improvement of Mongolian horse reproductive efficiency and breeding profitability. Therefore, there is an urgent need for a precise, efficient, and operable supplementary feeding technique that addresses the physiological and nutritional needs of late-pregnancy Mongolian horses during the winter and spring seasons, fully considering the differences between horses and other animals, as well as the unique characteristics of the Mongolian horse, to fill this technological gap and promote the healthy development of my country's horse industry. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a supplementary concentrate for Mongolian horses in late pregnancy. This supplementary concentrate is based on the special nutritional needs of mares in late pregnancy and takes into account the actual situation of the decline in the nutritional value of winter pasture in the Inner Mongolian grassland region. By scientifically quantifying the supplementary feed parameters, it ensures that mares receive comprehensive and balanced nutritional support.

[0007] Another object of the present invention is to provide a method for supplemental feeding of Mongolian horses in late pregnancy.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a supplementary concentrate for Mongolian horses in late pregnancy. The formula of the supplementary concentrate consists of the following ingredients in parts by weight: corn 70-72%, soybean meal 6-9%, wheat bran 8.7-16.2%, molasses 1-5%, vegetable oil 1-3%, limestone powder 0.5-1%, dicalcium phosphate 0.1-1%, lysine 0.1-1%, salt 0.1-1%, and premix 0.1-1%.

[0009] Preferably, the supplementary feed formula consists of the following ingredients in parts by weight: corn 71.4%, soybean meal 7.6%, wheat bran 8.7%, molasses 5.0%, vegetable oil 3.0%, limestone powder 0.8%, dicalcium phosphate 0.5%, lysine 1.0%, salt 1.0%, and premix 1.0%.

[0010] Preferably, the vegetable oil includes soybean oil, corn oil, or rapeseed oil.

[0011] Preferably, the 1% premix provides the following per kilogram of supplemental concentrate: 7000-8000 mg iron, 3000-4000 mg copper, 15000-20000 mg zinc, 4000-8000 mg manganese, 100-200 mg iodine, 40-70 mg selenium, 20-60 mg cobalt, 2000000-2200000 IU vitamin A, 15000-20000 IU vitamin E, and 3000000-3500000 IU vitamin D.

[0012] The present invention also provides a method for supplementing feeding Mongolian horses in late pregnancy, the method comprising the following steps: determining the daily amount of supplementary concentrate according to 0.6-0.8% of the mare's body weight, and feeding the determined supplementary concentrate to the mare.

[0013] Preferably, the supplemental concentrate is fed once a day at 7:00 AM.

[0014] Preferably, mare should be provided with free access to water and hay.

[0015] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in improving the growth performance or reproductive efficiency of Mongolian mare in late pregnancy.

[0016] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in enhancing the immune function or antioxidant capacity of Mongolian mare in late pregnancy.

[0017] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in maintaining the balance of intestinal microbiota in Mongolian mare during late pregnancy.

[0018] Compared with the prior art, the present invention has the following advantages: (1) This invention provides a supplementary concentrate and method for Mongolian horses in late pregnancy, aiming to solve key technical problems such as nutritional abortion, fetal maldevelopment, and decreased mare reproductive efficiency caused by the decline in the nutritional quality of natural pastures and the increased nutritional needs of mares in late pregnancy, especially in winter and early spring. Specifically, this invention systematically explores the effects of supplementary concentrates with different energy and protein levels on the growth performance, nutrient digestibility, blood biochemical indicators, reproductive hormone levels, antioxidant properties, immune function, and fecal microbiota and metabolomics of Mongolian horses in late pregnancy, thereby providing a theoretical basis and data support for determining a scientific supplementary diet for energy and protein in Mongolian horses in late pregnancy.

[0019] (2) Winters in the Inner Mongolian grasslands are long and cold, with natural pastures covered in snow, significantly reducing the nutritional value of forage. At this time, mares are in late pregnancy, with rapid fetal growth and development, and their nutritional needs reach their peak. Traditional grazing methods are insufficient to meet the nutritional needs of mares, leading to frequent problems such as nutritional abortion and poor fetal development. This invention effectively supplements the nutritional intake of mares during winter and early spring by providing supplemental concentrates with different energy and protein levels, ensuring that mares receive sufficient energy, protein, vitamins, and minerals to meet their needs for maintaining their health and for fetal growth and development.

[0020] (3) The reproductive efficiency of mares is directly related to the growth of the herd and the improvement of breed quality. Poor nutritional status of mares in late pregnancy not only affects the normal growth and development of the fetus, but also leads to difficulties in postpartum recovery, prolongs the reproductive cycle, and reduces reproductive efficiency. This invention improves the digestibility of nutrients in mares by scientifically supplementing them with concentrates, enhancing their immune function and antioxidant capacity, thereby improving their reproductive efficiency and the health of their offspring. At the same time, a suitable supplementary feeding program can also reduce the occurrence of postpartum complications in mares, shorten the breeding interval, and improve the overall reproductive performance of the herd.

[0021] (4) Gut microbiota play an important role in the digestion, absorption, immune regulation, and energy metabolism of mares. Changes in the nutritional status of mares during late pregnancy directly affect the composition and function of gut microbiota, thereby impacting the health of mares. This invention studies the effects of supplemental feed with different energy and protein levels on the gut microbiota of mares, screens out dominant microbiota beneficial to mare health, optimizes gut microbiota balance, and improves the digestive and absorptive efficiency and immune function of mares. Simultaneously, through metabolomics analysis, the complex relationship between gut microbiota and mare metabolism is revealed, providing a scientific basis for maintaining the gut health of mares.

[0022] (5) Winters are long and cold in northern my country, posing numerous challenges to the feeding and management of mares. This invention systematically studies the effects of supplemental feeding with concentrates of different energy and protein levels on Mongolian horses in late pregnancy, and screens out suitable supplemental feeding programs, providing scientific guidance for winter supplemental feeding of grazing mares in northern regions. Furthermore, the research findings of this invention can provide reference and guidance for the refined feeding and management of horses in other regions, promoting the transformation, upgrading, and sustainable development of the horse industry. Attached Figure Description

[0023] Figure 1 Design a flowchart for energy supplementation experiments; Figure 2 Design a flowchart for protein supplementation trials; Figure 3 The figure shows the α-diversity of microorganisms in mare feces under different energy levels. In the figure, A is the Chao1 index, B is the ACE index, C is the Shannon index, and D is the Sob index.

[0024] Figure 4 The figures show the characteristics of mare fecal microorganisms at the phylum and genus levels under different energy levels. Figure A shows the relative abundance distribution of the cecal microbial community at the phylum level, B shows the abundance differences at the specific phylum level, C shows the relative abundance distribution of the cecal microbial community at the genus level, and D shows the abundance differences at the specific genus level.

[0025] Figure 5 The figure shows the α-diversity of mare feces microorganisms under different protein levels. In the figure, A is the Shannon index and B is the Simpson index. Figure 6 The figures show the characteristics of fecal microorganisms in mare feces at the phylum and genus levels under different protein levels. In the figure, A represents the relative abundance distribution of the cecal microbial community at the phylum level, B represents the comparison of abundance differences at the specific phylum level, C represents the relative abundance distribution of the cecal microbial community at the genus level, and D represents the comparison of abundance differences at the specific genus level. Figure 7KEGG enrichment analysis of differential metabolites in mare feces at different energy levels. Figure A shows the KEGG enrichment analysis of differential metabolites between the low-energy group (LEN) and the medium-energy group (MEN); B shows the KEGG enrichment analysis of differential metabolites between the medium-energy group (MEN) and the high-energy group (HEN); and C shows the KEGG enrichment analysis of differential metabolites between the low-energy group (LEN) and the high-energy group (HEN).

[0026] Figure 8 KEGG enrichment analysis of differential metabolites in mare feces at different protein levels. Figure A shows the KEGG enrichment analysis of differential metabolites between the high protein group (HCP) and the low protein group (LCP); B shows the KEGG enrichment analysis of differential metabolites between the high protein group (HCP) and the medium protein group (MCP); and C shows the KEGG enrichment analysis of differential metabolites between the medium protein group (MCP) and the low protein group (LCP). Detailed Implementation

[0027] This invention provides a supplementary concentrated feed for Mongolian horses in late pregnancy. The preferred formulation of this supplementary concentrated feed consists of the following ingredients in parts by weight: corn 70-72%, soybean meal 6-9%, wheat bran 8.7-16.2%, molasses 1-5%, vegetable oil 1-3%, limestone powder 0.5-1%, dicalcium phosphate 0.1-1%, lysine 0.1-1%, salt 0.1-1%, and premix 0.1-1%. More preferably, the formulation consists of the following ingredients in parts by weight: corn 71.0-71.8%, soybean meal 6.9-8.5%, wheat bran 9.6-15.2%, and molasses 1%. The supplementary feed formula is further preferably composed of the following ingredients by weight: corn 71.4%, soybean meal 7.6%, wheat bran 8.7%, molasses 5.0%, vegetable oil 3.0%, limestone powder 0.8%, dicalcium phosphate 0.5%, lysine 1.0%, salt 1.0%, and premix 1.0%, with the total weight percentage of each ingredient in the supplementary feed being 100%. In this invention, the digestible energy of the supplementary feed is preferably 14-16 MJ / kg; the crude protein content of the supplementary feed is preferably 11-13%. More preferably, the digestible energy of the supplementary feed is 15.48 MJ / kg; and more preferably, the crude protein content of the supplementary feed is 12.04%. In this invention, the key nutritional indicators of the supplementary concentrate, such as digestible energy (DE), crude protein (CP), crude fat (EE), neutral detergent fiber (NDF), acid detergent fiber (ADF), calcium (Ca), and phosphorus (P), meet the nutritional needs of mares in late pregnancy. Experimental verification shows that this invention significantly improves the growth performance, enhances immune function, and maintains intestinal microbial balance in Mongolian mares by feeding them a medium-energy concentrate with 15.48 MJ / kg of digestible energy at 0.75% of their body weight, and / or a medium-protein concentrate with 12.04% crude protein at 0.75% of their body weight.

[0028] In this invention, the vegetable oil preferably includes soybean oil, corn oil, or rapeseed oil. As a preferred embodiment, the vegetable oil is selected from soybean oil. Soybean oil is rich in polyunsaturated fatty acids, especially linoleic acid (Omega-6). It is widely available, inexpensive, and has high energy value, helping to improve the digestible energy of the diet, making it more suitable as a basic energy supplement source.

[0029] In this invention, the 1% premix preferably provides per kilogram of supplementary concentrate: 7000-8000 mg iron, 3000-4000 mg copper, 15000-20000 mg zinc, 4000-8000 mg manganese, 100-200 mg iodine, 40-70 mg selenium, 20-60 mg cobalt, 2000000-2200000 IU vitamin A, 15000-20000 IU vitamin E, and 30000000-3500000 IU vitamin D; more preferably, the 1% premix provides per kilogram of supplementary concentrate: 7500 mg iron, 3600 mg copper, 18000 mg zinc, 6000 mg manganese, 120 mg iodine, 60 mg selenium, 60 mg cobalt, 2125000 IU vitamin A, 18750 IU vitamin E (tocopherol), and vitamin D. 3300000IU.

[0030] This invention also provides a method for supplementing the diet of Mongolian horses in late pregnancy. The method includes the following steps: determining the daily amount of supplementary concentrate based on 0.6-0.8% of the mare's body weight, and feeding the determined supplementary concentrate to the mare. Preferably, the daily amount of supplementary concentrate is determined to be 0.75% of the mare's body weight. This invention determines the daily amount of supplementary concentrate based on the mare's weight and nutritional needs, ensuring that the mare receives stable and continuous nutritional support.

[0031] In this invention, the supplemental concentrate is preferably fed once daily at 7:00 AM. This invention's choice of a single supplemental feeding at 7:00 AM comprehensively considers animal physiology, environmental climate, and feeding management. This invention fully aligns with the physiological rhythm of Mongolian horses' vigorous morning feeding in late pregnancy, providing concentrated and high-quality nutrition during the period when their digestive tract absorption efficiency is at its peak after a night of fasting, thereby maximizing nutrient utilization. Simultaneously, feeding at 7:00 AM effectively addresses the low morning temperatures of winter and spring, providing mares with immediate energy to resist cold stress and reduce the depletion of their reserves. Furthermore, this single, timed supplemental feeding not only avoids the burden of multiple feedings caused by fetal compression of the abdominal cavity, helping to maintain the mare's metabolic stability throughout the day, but also greatly simplifies daily farm management processes, facilitating centralized observation and precise monitoring, and forming a highly efficient nutritional complement to daytime roughage intake.

[0032] In this invention, during supplemental feeding, mares are ensured free access to water and hay, and a good feeding environment and management conditions are maintained to reduce the impact of stress factors on their health. This invention requires ensuring free access to water and hay for mares during supplemental feeding, which, together with a precise supplemental feeding program, constitutes a synergistic and complete nutritional system. Free access to water is a prerequisite for ensuring the full digestion, absorption, and utilization of all nutrients, while directly supporting pregnancy physiology, fetal development, and body temperature regulation; free access to hay maintains healthy peristalsis in the digestive tract through its physical fiber structure and provides a stable fermentation substrate for hindgut microorganisms.

[0033] In this invention, as an implementable method, indicators such as the weight, feed intake, health status and reproductive performance of mares are monitored regularly, and the supplementary feeding plan is adjusted in a timely manner based on the monitoring results to ensure the maximization of the supplementary feeding effect.

[0034] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in improving the growth performance or reproductive efficiency of Mongolian mare in late pregnancy.

[0035] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in enhancing the immune function or antioxidant capacity of Mongolian mare in late pregnancy.

[0036] The present invention also provides the application of the aforementioned supplementary concentrate or the aforementioned supplementary feeding method in maintaining the balance of intestinal microbiota in Mongolian mare during late pregnancy.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0039] Example 1 A supplementary concentrate for Mongolian horses in late pregnancy, the formula of which consists of the following ingredients by weight: corn 71.4%, soybean meal 7.6%, wheat bran 8.7%, molasses 5.0%, vegetable oil 3.0%, limestone powder 0.8%, dicalcium phosphate 0.5%, lysine 1.0%, salt 1.0%, and premix 1.0%; wherein the vegetable oil is soybean oil, and the 1.0% premix provides per kilogram of supplementary concentrate: 7500mg iron, 3600mg copper, 18000mg zinc, 6000mg manganese, 120mg iodine, 60mg selenium, 60mg cobalt, vitamin A 2125000IU, vitamin E (tocopherol) 18750IU, and vitamin D 3300000IU.

[0040] Example 2 A supplementary concentrate for Mongolian horses in late pregnancy, the formula of which consists of the following ingredients in parts by weight: corn 70.0%, soybean meal 9.0%, wheat bran 12.5%, molasses 5.0%, vegetable oil 1.0%, limestone powder 0.5%, dicalcium phosphate 0.1%, lysine 0.8%, salt 0.1%, and premix 1.0%; wherein the vegetable oil is corn oil, and the 1.0% premix provides per kilogram of supplementary concentrate: 7500mg iron, 3600mg copper, 18000mg zinc, 6000mg manganese, 120mg iodine, 60mg selenium, 60mg cobalt, 2125000IU vitamin A, 18750IU vitamin E (tocopherol), and 3300000IU vitamin D.

[0041] Example 3 A supplementary concentrate for Mongolian horses in late pregnancy, the formula of which consists of the following ingredients by weight: 72% corn, 6% soybean meal, 16.2% wheat bran, 1.0% molasses, 1.5% vegetable oil, 1.0% limestone powder, 1.0% dicalcium phosphate, 0.1% lysine, 0.2% salt, and 1.0% premix; wherein the vegetable oil is rapeseed oil, and the 1.0% premix provides per kilogram of supplementary concentrate: 7500mg iron, 3600mg copper, 18000mg zinc, 6000mg manganese, 120mg iodine, 60mg selenium, 60mg cobalt, 2125000IU vitamin A, 18750IU vitamin E (tocopherol), and 3300000IU vitamin D.

[0042] Example 4 A method for supplementary feeding of Mongolian horses in late pregnancy, the steps of which are as follows: Supplementary feeding amount and frequency: Based on the mare's weight and nutritional needs, determine the daily supplementary feed amount of Example 1 at 0.75% of the mare's body weight, and feed it once a day at 7:00 am to ensure that the mare receives stable and continuous nutritional support. Feeding and management: During supplementary feeding, ensure that mares have free access to water and hay, and maintain a good feeding environment and management conditions to reduce the impact of stress factors on the health of mares.

[0043] Example 5 A method for supplementary feeding of Mongolian horses in late pregnancy, the steps of which are as follows: Supplementary feeding amount and frequency: Based on the mare's weight and nutritional needs, determine the daily supplementary feed amount of Example 2 at 0.75% of the mare's body weight, and feed it once a day at 7:00 am to ensure that the mare receives stable and continuous nutritional support. Feeding and management: During supplementary feeding, ensure that mares have free access to water and hay, and maintain a good feeding environment and management conditions to reduce the impact of stress factors on the health of mares.

[0044] Example 6 A method for supplementary feeding of Mongolian horses in late pregnancy, the steps of which are as follows: Supplementary feeding amount and frequency: Based on the mare's weight and nutritional needs, determine the daily supplementary feed amount of Example 3 at 0.75% of the mare's body weight, and feed it once a day at 7:00 am to ensure that the mare receives stable and continuous nutritional support. Feeding and management: During supplementary feeding, ensure that mares have free access to water and hay, and maintain a good feeding environment and management conditions to reduce the impact of stress factors on the health of mares.

[0045] Experimental Example 1 1. Selection and grouping of mares: Mongolian horses in late pregnancy with similar age, weight, parity, and gestation period were selected as experimental subjects. The selected mares were randomly divided into three groups, each containing 6 Mongolian horse mares, as low-energy / low-protein (LEN / LCP), medium-energy / medium-protein (MEN / MCP), and high-energy / high-protein (HEN / HCP) supplementary feeding groups, respectively.

[0046] 2. Supplemental concentrate formulation design: Based on the nutritional needs of mares in late pregnancy, supplementary concentrate formulas with different energy and protein levels are designed. The basic components of the supplementary concentrate include corn, soybean meal, and wheat bran, and different energy and protein levels are achieved by adjusting the proportions of these components.

[0047] Ensure that the nutritional components (such as vitamins and minerals) other than energy and protein in the supplemental feed of each group are consistent to eliminate interference from other factors.

[0048] Energy supplementation levels: Based on the mares' weight, parity, and gestation stage, three energy levels were established for supplementation: low energy level (LEN group, 13.93 MJ / kg DE), medium energy level (MEN group, 15.48 MJ / kg DE), and high energy level (HEN group, 16.95 MJ / kg DE), as detailed in Table 1. Dietary composition and nutrient levels are shown in Tables 2 and 3.

[0049] Table 1. Composition and nutrient levels of the supplemental concentrate (DM, %) in the experiment

[0050] Note: 1% premix provides the following per kilogram of supplemental concentrate: 7500 mg iron, 3600 mg copper, 18000 mg zinc, 6000 mg manganese, 120 mg iodine, 60 mg selenium (of which 15 mg is organic selenium), 60 mg cobalt, 2125000 IU vitamin A, 18750 IU vitamin E, and 3300000 IU vitamin D.

[0051] Table 2 Dietary Composition

[0052] Table 3 Nutritional levels of hay

[0053] Protein supplementation levels: To address the specific protein requirements of mares in late pregnancy, three protein levels were established for supplementation: low protein (LCP group, 10.85% CP), medium protein (MCP group, 12.04% CP), and high protein (HCP group, 13.25% CP), as detailed in Table 4. Dietary composition and nutrient levels are shown in Tables 5 and 6.

[0054] Table 4. Composition and nutritional levels of the supplemental concentrate (DM, %) in the experiment

[0055] Note: 1% premix provides the following per kilogram of supplemental concentrate: 7500 mg iron, 3600 mg copper, 18000 mg zinc, 6000 mg manganese, 120 mg iodine, 60 mg selenium (of which 15 mg is organic selenium), 60 mg cobalt, 2125000 IU vitamin A, 18750 IU vitamin E, and 3300000 IU vitamin D.

[0056] Table 5. Composition of Dietary Rations

[0057] Table 6 Nutritional Levels of Hay

[0058] 3. Setting the amount and frequency of supplementary feeding: Based on the mare's weight and nutritional needs, calculate the daily supplementary concentrate amount at 0.75% of the mare's body weight. Feed the determined daily supplementary concentrate once a day at 7:00 AM to ensure that the mare receives stable and continuous nutritional support.

[0059] 4. Feeding and Management: (1) During the supplementary feeding period, ensure that the mares have free access to water and provide sufficient clean drinking water.

[0060] (2) Free access to hay ensures that mares have enough roughage to maintain normal digestive function.

[0061] (3) Maintain a good feeding environment and management conditions, clean the pens regularly, and reduce the impact of stress factors on the health of mares.

[0062] 5. Data monitoring and recording: (1) Regularly monitor and record indicators such as the weight, feed intake, health status and reproductive performance of mares.

[0063] (2) Collect blood and fecal samples from mares for subsequent analysis of blood biochemical indicators, reproductive hormone levels, antioxidant properties, immune function and fecal microbiota.

[0064] 6. Results Analysis (1) Growth performance a. Final weight: As shown in Table 7, the final weight of mares in the MEN group (medium energy level) was significantly higher than that in the HEN group (high energy level) and the LEN group (low energy level), with a P value of 0.014, indicating that the medium energy level has a significant promoting effect on the weight gain of mares.

[0065] b. Daily weight gain As shown in Table 8, the daily weight gain of mares in the MCP group was significantly higher than that in the HCP group (high protein group), P<0.05, indicating that the medium energy level has a positive effect on the daily weight gain of mares.

[0066] Table 7. Effects of different energy levels of supplemental concentrate feeding on growth performance of Mongolian horses in late pregnancy.

[0067] Note: The absence of identical letters in the same row's shoulder label indicates a significant difference (P < 0.05).

[0068] Table 8. Effects of different protein levels in supplemental concentrate feed on growth performance of mares in late pregnancy.

[0069] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0070] (2) Nutrient digestibility According to the results in Table 9, the apparent digestibility of CP in mares in the LEN group was significantly higher than that in the MEN group (P=0.022), while the HEN group showed no significant difference compared to the other two groups. For DM, the apparent digestibility differed significantly among the three groups, with the MEN group having the highest, followed by the LEN group, and the HEN group having the lowest (P<0.001). The apparent digestibility of OM and Ca in mares in the MEN group was significantly higher than that in the HEN and LEN groups (P=0.013, P<0.001), while there was no significant difference between the HEN and LEN groups. For ADF, the apparent digestibility differed significantly among the three groups, with the MEN group having the highest, followed by the HEN group, and the LEN group having the lowest (P<0.001).

[0071] The results in Table 10 show that the CP (Cost Per Count) in the HCP group was significantly higher than that in the LCP group (P=0.027). The EE (Excretion Efficacy) digestibility in the HCP group was significantly lower than that in the MCP and LCP groups (P<0.001). The DM (Desmodium Dioxide) digestibility in the HCP group was significantly lower than that in the MCP and LCP groups (P<0.001). The OM (Oxygen Deficiency) digestibility was highest in the MCP group, significantly higher than that in the LCP and HCP groups (P<0.001). The P (Potassium Dioxide) digestibility in the HCP group was significantly lower than that in the MCP and LCP groups (P<0.001). The NDF (Nutrient Detoxification) digestibility in the MCP group was significantly higher than that in the LCP and HCP groups (P=0.020).

[0072] Table 9. Effects of different energy levels of supplemental concentrate feeding on apparent nutrient digestibility in late-pregnancy Mongolian horses.

[0073] Note: In the table, the same letter in the same superscript indicates no significant difference, and different letters in the superscript indicate a significant difference. P <0.05).

[0074] Table 10. Effects of different protein levels in supplemental concentrate on apparent nutrient digestibility in late-gestation Mongolian horses.

[0075] Note: In the table, the same letter in the same superscript indicates no significant difference, and different letters in the superscript indicate a significant difference. P <0.05).

[0076] (3) Blood biochemical indicators and reproductive hormones As shown in Table 11, lactate dehydrogenase (LDH) activity was significantly higher in the MEN group than in the HEN and LEN groups (P=0.001). Conversely, aspartate aminotransferase (AST) activity was significantly lower in the MEN group than in the HEN and LEN groups (P=0.009). The differences between groups for other indicators did not reach statistical significance.

[0077] As shown in Table 12, the creatinine (CREA) level in the MCP group was significantly lower than that in the LCP and HCP groups (P<0.001). The creatine kinase (CK) level in the HCP group was significantly higher than that in the LCP and MCP groups (P<0.001). The lactate dehydrogenase (LDH) level in the HCP group was significantly higher than that in the MCP and LCP groups (P<0.001). The aspartate aminotransferase (AST) level in the MCP group was significantly higher than that in the LCP and HCP groups (P<0.001).

[0078] Table 11 Effects of different energy levels of supplemental concentrate feeding on serum biochemical parameters in Mongolian horses during late pregnancy.

[0079] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0080] Table 12 Effects of different protein levels in supplemental concentrate on serum biochemical parameters in Mongolian horses during late pregnancy.

[0081] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0082] (4) Antioxidant properties and immune function Table 13 shows that the gonadotropin-releasing hormone (GnRH) levels were significantly different among the three groups (P<0.001), with the highest GnRH level in the LEN group, followed by the HEN group, and the lowest in the MEN group. Follicle-stimulating hormone (FSH) and oxytocin (OXT) levels were significantly higher in the LEN group than in the HEN group (P=0.02, P=0.013). Furthermore, estradiol (E2) levels were significantly higher in the LEN group than in the MEN and HEN groups (P=0.002). For prolactin (PRL) and progesterone (P4) levels, there were also significantly different levels among the three groups (P<0.001, P<0.001), with the highest in the LEN group, followed by the MEN group, and the lowest in the HEN group. Testosterone (T) levels were significantly lower in the HEN group than in the LEN and MEN groups (P=0.018).

[0083] Table 14 shows that E2 in the MCP and HCP groups was significantly higher than that in the LCP group (P=0.005). LH in the HCP group was significantly higher than that in the LCP and MCP groups (P<0.001). P4 in the HCP group was extremely significantly higher than that in the MCP and LCP groups (P<0.001). FSH in the HCP group was extremely significantly higher than that in the MCP and LCP groups (P<0.001). PRL in the HCP group was significantly higher than that in the MCP and LCP groups (P<0.001). GnRH in the HCP group was also significantly higher than that in the MCP and LCP groups.

[0084] Table 13 Effects of different energy levels of supplemental concentrate on serum reproductive hormones in Mongolian horses during late pregnancy.

[0085] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0086] Table 14 Effects of different protein levels in supplemental concentrate on serum reproductive hormones in Mongolian horses during late pregnancy.

[0087] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0088] Table 15 shows that different energy levels of concentrate significantly affected the antioxidant capacity of Mongolian horses in late pregnancy. Glutathione peroxidase (GSH-Px) activity was significantly lower in the HEN group than in the LEN and MEN groups (P=0.01). Catalase (CAT) content showed highly significant differences among the three groups (P<0.001), with the highest in the MEN group, followed by the LEN group, and the lowest in the HEN group. Furthermore, malondialdehyde (MDA) content also showed highly significant differences among the three groups (P<0.001), with the highest in the HEN group, followed by the LEN group, and the lowest in the MEN group. Superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) showed no significant differences among the three groups (P>0.05).

[0089] As shown in Table 16, the GSH-Px activity in the LCP group was significantly higher than that in the MCP and HCP groups (P<0.001). The CAT activity in the MCP group was significantly higher than that in the LCP and HCP groups (P<0.001). The MDA activity in the HCP group was significantly higher than that in the LCP and MCP groups (P<0.001).

[0090] Table 15 Effects of different energy levels of supplemental concentrate feeding on serum antioxidant indices in Mongolian horses during late pregnancy.

[0091] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0092] Table 16 Effects of different protein levels in supplemental concentrate feed on serum antioxidant indices in Mongolian horses during late pregnancy.

[0093] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0094] Table 17 shows that different energy levels of concentrate significantly affected the immune function of Mongolian horses in late pregnancy. The levels of immunoglobulin M (IgM) and interleukin-1β (IL-1β) were significantly lower in the HEN group than in the LEN and MEN groups (P=0.021, P<0.001). Conversely, the levels of immunoglobulin A (IgA) and immunoglobulin G (IgG) were significantly higher in the MEN group than in the LEN and HEN groups (P<0.001, P=0.049). Furthermore, the level of interleukin-2 (IL-2) was significantly higher in the HEN group than in the LEN and MEN groups (P<0.001).

[0095] Table 18 shows that IgM in the LCP group was significantly higher than that in the MCP and HCP groups (P<0.001). IgA in the LCP group was significantly higher than that in the MCP and HCP groups (P<0.001). IL-1β in the MCP group was significantly higher than that in the LCP and HCP groups (P<0.001), while the HCP group had the lowest IL-6. IL-6 in the LCP and HCP groups was significantly higher than that in the MCP group (P<0.001). TNF-α in the HCP group was significantly higher than that in the LCP and MCP groups (P=0.025).

[0096] Table 17 Effects of different energy levels of supplemental concentrate feeding on serum immune markers in Mongolian horses during late pregnancy.

[0097] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0098] Table 18 Effects of different protein levels in supplemental concentrate feeding on serum immune markers in Mongolian horses during late pregnancy.

[0099] Note: If the same letter is used in the same row of the table, the difference is not significant; if the letters are different, the difference is significant (P<0.05).

[0100] (5) Fecal microbiota and metabolomics a. Microbial diversity: The MEN group was able to increase the abundance of beneficial gut bacteria. Among the Chao1, ACE, Shannon, and Simpson indices, the MEN group had the highest mean diversity. Figure 3 Based on the results of microbial taxonomy analysis, at the phylum level ( Figure 4 A) A total of 11 phylum-level taxonomic units were identified, among which Firmicutes, Proteobacteria, Bacteroidetes, and Verrucomicrobiota were the dominant phyla, accounting for more than 80% in total. Specifically, Firmicutes and Proteobacteria were the most prevalent, with significantly higher relative abundance than other phyla. Intergroup comparisons revealed that the relative abundance of Firmicutes in the MEN group was significantly higher than that in the LEN group (P < 0.05), while the relative abundance of Proteobacteria in the LEN group was significantly higher than that in the MEN group (P < 0.05). Figure 4 B). At the genus level ( Figure 4C) A total of 11 genus-level taxonomic units were identified, among which *Acinetobacter*, *Solibacillus*, and the Rikenellaceae RC9 gut group were the dominant families. The relative abundance of *Acinetobacter* in the LEN group was significantly higher than that in the MEN group (P < 0.05), while the relative abundance of *Rummeliibacillus*, *Anaerovorax*, and *Oribacterium* in the MEN group was significantly higher than that in the LEN group (P < 0.05). Figure 4 D).

[0101] The MCP group was able to increase the abundance of beneficial gut bacteria. Among the Shannon and Simpson indices, the MCP group had the highest average diversity, which was significantly greater than that of the LCP group (P<0.05). Figure 5 Gate-level analysis () Figure 6 A) showed that the cecal microbiota was mainly composed of Firmicutes and Bacteroidetes, which together accounted for 80%–90% of the total microbiota. In addition, Proteobacteria and Actinobacteria were also present in small amounts. Proteobacteria were significantly higher in the HCP group than in the MCP and LCP groups (P<0.01), while there was no significant difference between the MCP and LCP groups (P>0.05). The LCP group had the highest proportion of Bacteroidetes (38%), significantly higher than the HCP and MCP groups (P<0.05). The abundance of soluble bacteria (Patescibacteria) was significantly higher in the MCP group than in the LCP group (P<0.05), while there was no significant difference in abundance between the MCP and HCP groups (P>0.05). Figure 6 B).

[0102] genus level analysis ( Figure 6C) showed that *Prevotella* and *Ruminococcus* were the dominant genera, accounting for 30%-40% of the total flora. The abundance of *Acinetobacter* in the HCP group was significantly higher than that in the MCP and LCP groups (P<0.01), while there was no significant difference between the MCP and LCP groups (P>0.05). The abundance of the *Rikenellaceae_RC9_gut_group* in the MCP group was significantly higher than that in the LCP and HCP groups (P<0.01), while there was no significant difference between the HCP and LCP groups (P>0.05). The abundance of *Rummeliibacillus* in the LCP group was the lowest, significantly lower than that in the MCP group (P<0.05), but there was no significant difference compared to the HCP group (P>0.05). The LCP group had the lowest abundance of NK4A214 group, significantly lower than the MCP group (P<0.05), but no significant difference compared with the HCP group (P>0.05). Figure 6 D).

[0103] b. Differential metabolites: according to Figure 7 The results showed that the differentially metabolite KEGG in the feces of the MEN group was mainly enriched in signaling pathways related to carbohydrate digestion and absorption, insulin secretion, fatty acid degradation, oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, vitamin digestion and absorption, and glycolysis / gluconeogenesis. This indicates that supplementing with medium-energy concentrate significantly altered the composition, structure, and function of the fecal microbiota in late-pregnancy mares, leading to differential enrichment of specific bacterial species. With these changes in the fecal microbiota and its metabolites, the mares' nutritional metabolism and overall health are likely to improve.

[0104] KEGG enrichment analysis was performed on differentially expressed metabolites from the LCP, MCP, and HCP groups. The top 10 significantly enriched signaling pathways in each group were selected (P<0.05). Among the protein-related pathways in the HCP group versus the LCP group, lysine degradation, histidine metabolism, fatty acid degradation, and steroid hormone biosynthesis were found to be significantly enriched. Figure 8 A). Protein-related pathways in the HCP group vs. MCP group include the AMPK signaling pathway and D-amino acid metabolism. Figure 8B. In the MCP vs. LCP groups, protein-related pathways included lysine degradation, arginine and proline metabolism, and tryptophan metabolism. All three groups were enriched in phenylalanine, tyrosine, and tryptophan biosynthesis. Figure 8 C).

[0105] The data above indicate that, at medium energy levels (15.48 MJ / kg DE) and medium protein levels (12.04% CP), mares exhibited optimal performance in growth performance, nutrient digestibility, blood biochemical indicators and reproductive hormones, antioxidant capacity and immune function, as well as fecal microbiota and metabolomics. This invention provides a precise supplemental concentrate and a corresponding method, offering a complete solution for the nutritional management of Mongolian horses in late pregnancy. This technical solution is mature enough for commercialization, enabling the preparation of standardized and regulated premixed feeds or complete concentrate supplements based on the described formula and process. The widespread application of this product will help improve mare reproductive efficiency, ensure offspring health, and optimize pasture management, thereby providing direct technical support and product assurance for the transformation, upgrading, and development of the horse industry in northern my country's pastoral areas.

[0106] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A supplementary concentrate for Mongolian horses in late pregnancy, characterized in that, The supplementary concentrate formula consists of the following ingredients by weight: corn 71.4%, soybean meal 7.6%, wheat bran 8.7%, molasses 5.0%, vegetable oil 3.0%, limestone powder 0.8%, dicalcium phosphate 0.5%, lysine 1.0%, salt 1.0%, and premix 1.0%. The vegetable oils include soybean oil, corn oil, or rapeseed oil; The 1% premix provides the following per kilogram of supplemental concentrate: 7000-8000 mg iron, 3000-4000 mg copper, 15000-20000 mg zinc, 4000-8000 mg manganese, 100-200 mg iodine, 40-70 mg selenium, 20-60 mg cobalt, 2000000-2200000 IU vitamin A, 15000-20000 IU vitamin E, and 3000000-3500000 IU vitamin D.

2. The supplemental concentrate according to claim 1, characterized in that, The supplemental concentrate was fed once a day at 7:00 AM.

3. The application of the supplemental concentrate according to any one of claims 1-2 in the preparation of diets to improve the growth performance or reproductive efficiency of Mongolian mares in late pregnancy, characterized in that, The diet consists of hay and supplemental concentrate, with the daily amount of supplemental concentrate determined according to 0.6-0.8% of the mare's body weight.

4. The application of the supplemental concentrate according to any one of claims 1-2 in the preparation of diets that enhance the immune function or antioxidant capacity of Mongolian mares in late pregnancy, characterized in that... The diet consists of hay and supplemental concentrate, with the daily amount of supplemental concentrate determined according to 0.6-0.8% of the mare's body weight.

5. The application of the supplemental concentrate according to any one of claims 1-2 in the preparation of a diet for maintaining the balance of intestinal microbiota in Mongolian mares during late pregnancy, characterized in that... The diet consists of hay and supplemental concentrate, with the daily amount of supplemental concentrate determined according to 0.6-0.8% of the mare's body weight.

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