Supplementary feeding method for yak hay in high and cold grassland withering period by adding rumen bypass glucose

Abstract

The invention discloses a supplementary feeding method for yak hay in a high and cold grassland withering period by adding rumen bypass glucose. The supplementary feeding method comprises the following steps: S1, selecting a perinatal-period plateau yak; s2, staged feeding management is conducted on the plateau yaks in the perinatal period, feeding is started 14 days before the perinatal period, feeding is conducted in a 7-day pre-feeding period and a 42-day positive feeding period, the pre-feeding period is used for establishing a feeding rhythm, and the nutrition metabolism indexes of the yaks are continuously monitored in the positive feeding period; feeding in different time periods every day: feeding mixed hay containing rumen bypass glucose in the breeding house within the time period of 9: 00-10: 30 in the morning; in the time period of 17: 00-18: 30 in the afternoon, independently feeding hay without rumen bypass glucose into the breeding house; after breeding house feeding is completed, the yaks are driven to the peripheral flat dam area for free feeding and water drinking. According to the invention, the rumen bypass glucose is added into the hay for supplementary feeding of the yaks, so that the cost is reduced, and the female yaks in the calving period are supplemented with the rumen bypass glucose added into the hay, so that the body health of the calves can be facilitated, and the occurrence of nutrition metabolism diseases is reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of feed additives, and particularly relates to a method for supplementally feeding highland cattle with dry grass added with rumen bypass glucose in the dry season of high-cold grassland. BACKGROUND

[0002] Since the perinatal period of yaks is generally in March-April, which is the period of extreme shortage of pasture in plateau pasturing areas, and yaks are generally raised on the plateau, the altitude is high, the natural environment is harsh, the weather is bad, the grassland is degraded, and the pasture is scarce, and many factors lead to insufficient intake of nutrients by yaks in the perinatal period, thereby causing insufficient energy supply of yaks in the perinatal period to meet the rapid growth of fetuses and lactation. Since yaks are large in size and fierce and combative in nature, it is difficult for people to control them, and if glucose is directly injected intravenously or infused into the gastrointestinal tract, the yaks will be injured, and it will also be dangerous to personnel. Therefore, there is an urgent need for a way of feeding to directly deliver glucose to the small intestine to provide energy for yaks in the perinatal period and for milk production.

[0003] The existing document “Chao Wenju. Effects of supplement feeding on production performance of perinatal yaks and growth and development of their calves [D]. Qinghai University, 2009” discloses effects of supplement feeding on production performance of perinatal yaks and growth and development of their calves, and the feeding of yaks is realized by separately feeding each yak with concentrate and oat grass, and then allowing the yaks to freely lick the lick brick (urea molasses lick brick). The supplement feeding has a great influence on part of the production performance of perinatal yaks, and improves the milk yield of yaks, but the concentrate, oat grass and lick brick are basically absorbed after passing through the stomach of yaks, and are rarely absorbed in the small intestine, resulting in insufficient intake of energy by yaks. And the addition of urea molasses in the lick brick increases the cost.

[0004] Existing technologies include rumen-protected glucose, which, through special coating or processing, effectively resists fermentation and degradation by rumen microorganisms, allowing it to pass smoothly through the rumen to the small intestine where it is broken down and absorbed by digestive enzymes. However, the effects of different supplemental feeding dosages and timings on the long-term growth and reproductive performance of calves are not fully understood. Current research focuses primarily on short-term growth performance assessments, while systematic studies on the long-term effects of rumen-protected glucose throughout the calf's growth cycle, especially its potential impact on reproductive performance, are still lacking. Secondly, the synergistic mechanism between rumen-protected glucose and other nutrients requires further investigation. Adding rumen-protected glucose to concentrate feed increases costs for herders, as they primarily rely on grazing for their livelihood and generally have limited economic means. Feeding yaks entirely with concentrate feed would create an economic burden for herders, and the transportation costs of concentrate feed are also high. Furthermore, the compression process during lick block production means that directly adding rumen-protected glucose to the feed could cause the outer coating of the glucose to rupture, preventing its absorption in the small intestine. Summary of the Invention

[0005] The purpose of this invention is to solve the problem of insufficient energy during the perinatal period; at the same time, it avoids the defects of traditional brick-licking methods that squeeze and damage the cladding layer, thereby reducing the economic burden on herders.

[0006] To achieve the above objectives, this invention provides a method for supplementing yak hay with rumen-treated glucose during the dry season in alpine grasslands. After weighing out the required glucose dosage for this experiment, the hay and glucose are placed in a basin and stirred thoroughly. Water is added during the stirring process to ensure the rumen-treated glucose adheres better to the hay, guaranteeing sufficient mixing. Each yak uses its own basin. After feeding, the basins are cleaned. To prevent significant deviations in the glucose dosage during subsequent feedings, the specific steps are as follows: S1. Select 30 peripartum plateau yaks weighing 247-287kg, aged 3.5 years and in good health, and divide them into 3 groups of 10 each; S2, Staged Feeding Management 0. Yaks are fed approximately 14 days before the birthing period, with a 7-day pre-feeding period and a 42-day regular feeding period. The 7-day pre-feeding period is used to establish the feeding rhythm, and the 42-day regular feeding period involves continuous monitoring of nutritional and metabolic indicators.

[0007] 1. Implement staggered feeding times daily: From 9:00 to 10:30 AM, feed hay containing rumen glucose in the barn to ensure the yaks ingest rumen glucose in one go; from 5:00 to 6:30 PM, feed only hay in the barn. The hay containing rumen glucose is prepared by placing 150g of rumen glucose and 300g of hay in a special feeding container, adding 50ml of water and stirring to ensure the glucose adheres evenly to the surface of the hay.

[0008] c. After feeding the yaks in the pen, drive them to the outer flat area for free grazing and drinking (there is a flat area outside each pen. During the experiment, the hay was fed inside the pen to ensure that the yaks could finish the rumen glucose. However, the amount of hay fed was not enough to meet the needs of the yaks. So after feeding the rumen glucose inside the pen, the yaks were driven to the flat area and then fed hay).

[0009] Beneficial effects: 1. In this invention, by adding rumen-exposed glucose to hay to supplement yak feed, the problem of increased costs caused by directly adding rumen-exposed glucose to licks is solved. Therefore, adding rumen-exposed glucose directly to hay simplifies the experiment and reduces costs.

[0010] 2. Adding 150g of rumen-exposed glucose to the hay supplemented during the calving period of yaks can significantly improve calf growth performance. Adding 150g of rumen-exposed glucose to the diet can significantly increase lactose, milk fat, milk protein, and energy in milk. Adding 150g of rumen-exposed glucose to hay can significantly reduce the levels of β-hydroxybutyrate, serum total cholesterol, albumin, and triglycerides in calves, and significantly increase serum glucose levels. Adding rumen-exposed glucose to calving female yaks can benefit the health of calves and reduce the incidence of nutritional metabolic diseases. Attached Figure Description

[0011] Figure 1 This is a graph showing the effect of feeding RPG to yaks during the peripartum period on growth-related hormone indicators in calves, provided by the present invention. Figure 2 This is a graph showing the effect of feeding RPG to yaks during the peripartum period on relevant technical indicators of calves, provided by the present invention. Figure 3 This is a graph showing the effect of feeding RPG to yaks during the peripartum period on routine serum indicators in calves, provided by the present invention. Figure 4 This is a graph showing the effect of feeding RPG to yaks during the peripartum period on the immune performance of calves, provided by the present invention. Figure 5 This is a graph showing the effect of feeding RPG to yaks during the peripartum period on the antioxidant properties of calves, provided by the present invention. Figure 6 This is a dilution curve of Sobs in calf feces provided by the present invention; Figure 7 This is a Shannon dilution curve of calf feces microorganisms provided by the present invention; Figure 8 This is a VEN diagram of calf feces microorganisms provided by the present invention; Figure 9 This is a diagram of the microbial species composition of calf manure provided by the present invention; Figure 10 This is a VEN diagram comparing samples from different groups of calf serum metabolomes provided by the present invention; Figure 11 This is a KEGG pathway enrichment analysis diagram of calf serum metabolome provided by the present invention; Figure 12 This is a differential abundance score map of the KEGG pathway in calf serum metabolome provided by the present invention. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0013] The application principle of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0014] Explanation of related meaning: Rumen-protected glucose (RPG) is a good source of glucose for alleviating negative energy balance (NEB) in early lactation dairy cows. It is encapsulated in hydrogenated fats to evade rumen digestion and is completely released upon entering the intestines. Therefore, more glucose is transported to the small intestine and absorbed directly by the intestinal epithelium, rather than relying entirely on hepatic gluconeogenesis.

[0015] The mechanism of action of rumen-protected glucose is mainly reflected in the following aspects: First, through a special coating process, rumen-protected glucose effectively avoids fermentation and degradation in the rumen, allowing it to be directly absorbed and utilized in the small intestine, significantly improving glucose utilization efficiency and providing animals with a stable energy source. Second, after entering the small intestine, rumen-protected glucose can rapidly increase blood glucose levels, stimulate insulin secretion, thereby promoting cellular glucose uptake and utilization, maintaining stable blood glucose levels, and inhibiting lipolysis and gluconeogenesis, reducing energy waste. Furthermore, rumen-protected glucose can improve intestinal mucosal structure, enhance the intestine's ability to digest and absorb nutrients, and promote healthy intestinal development. In terms of immune function, rumen-protected glucose can significantly increase the content of immunoglobulins in the blood of calves, enhance humoral immunity, and reduce morbidity and mortality during the growth process.

[0016] Example 1: The experiment was conducted at the Yak Farm of the Grassland Science Research Institute in Hongyuan County, Aba Tibetan and Qiang Autonomous Prefecture, Sichuan Province, at an average altitude of 3500 meters. This example study investigated the effects of rumen-treated glucose supplementation on calf growth performance and its underlying mechanisms in peripartum yaks. Details are as follows:

[0017] (1) Select 30 peripartum plateau yaks with similar weight (247~287kg), age 3.5 years and good health, and divide them into 3 groups of 10 each. The pre-feeding period is 7 days and the regular feeding period is 42 days. During the experiment, keep the barn clean, clean the barn regularly, and allow the cows free access to water.

[0018] (2) Feeding time: From 9:00 to 10:30 in the morning, hay containing rumen glucose was fed in the pen. From 17:00 to 18:30, only hay was fed in the pen. After feeding the yaks in the pen, the yaks were driven to the outer plain area for free grazing and drinking. The first stage of the experiment ended when the cows began to calve. The three groups were designed as the control group, experimental group 1, and experimental group 2, respectively.

[0019] The control group was fed only hay as usual; Experimental group 1: 150g of rumen-exposed glucose (low RPG group) was added to 300g of hay, and 50ml of water was added and stirred to make the glucose evenly adhere to the surface of the hay. Experimental group 2: 300g of rumen-exposed glucose (high RPG group) was added to 500g of hay, and 75ml of water was added and stirred. (3) After the cows gave birth, 18 calves with similar weight, good health and similar birth dates were selected and divided into 3 groups of 6. The calves were recorded and numbered according to the cows they gave birth to. The first sampling was carried out 7 days after birth, and then the sampling was carried out at 7-day intervals. A total of four samples were collected. During this period, the calves ate and drank normally.

[0020] (4) On the 7th, 14th, 21st and 28th day after birth, before the morning feeding, about 10 ml of blood was collected from the calf using the jugular vein blood collection method. The blood was collected using a non-anticoagulated vacuum blood collection tube, left to stand for 1 hour, and centrifuged at 3500xg for 10 min. The supernatant was separated and stored at -20℃ for later use. On the 14th day before the morning feeding, about 50 ml of normal milk was collected from the mother yak, packaged and stored at -20℃ for later use.

[0021] The experimental data were statistically analyzed and organized using Excel. All data were analyzed using one-way ANOVA with GraphPad, and the results are presented in bar charts. * indicates a significant difference (P < 0.05), and ** and above indicate an extremely significant difference (P < 0.01). In the same row of data, different lowercase letters in the superscript indicate a significant difference (P < 0.05), while identical lowercase letters or no letters indicate no significant difference (P > 0.05).

[0022] Determination of milk component indicators: Before analysis, the frozen milk sample was thawed in a 40℃ water bath and gently mixed to ensure uniform temperature. The milk protein was determined using a fully automatic Kjeldahl nitrogen analyzer (VAP 450), the milk fat was determined using a fat analyzer (YSP 191), the ash was determined using a box-type resistance furnace (SX2-8-10), the lactose was determined using a liquid chromatograph (Ultimate 3000), and the moisture was determined using an electric heating drying oven (101-3AB).

[0023] Analysis of normal milk collected during the RPG feeding trial period revealed the following (Table 1): The concentrations of energy, lactose, milk fat, and milk protein in the low-RPG group were significantly higher than those in the control and high-RPG groups (P < 0.05), while the concentrations of moisture, ash, and non-fat milk solids showed no significant differences (P > 0.05). The significantly higher lactose, energy, milk protein, and milk fat content in the milk of the low-RPG group compared to the control group indicates that feeding peripartum yaks with low-dose RPG significantly increases milk nutritional composition, providing sufficient energy for calf growth and improving their growth performance.

[0024] Table 1. Effects of rumen-treated glucose supplementation on milk composition in periparturient yaks Determination of calf growth performance indicators: The weight, body size, height, and chest circumference of calves were recorded at 1 day, 14 days, 21 days, and 28 days after birth, before the cow's morning feeding.

[0025] Table 2 shows that the birth weight of calves fed with low RPG was significantly higher than that of the control and high RPG groups (P < 0.01), and the data at 14, 21, and 28 days were also significantly higher in the low RPG group than in the control group (P < 0.05). Regarding body size, the birth body size of the low RPG and control groups was significantly higher than that of the high RPG group (P < 0.05), and the measurements of the low RPG group at 14, 21, and 28 days were also significantly higher than those of the control and high RPG groups. However, the body size (body oblique length) of the control group was significantly higher than that of the low RPG group at 14 days (P < 0.05). There were no significant differences in chest circumference and body height.

[0026] Table 2. Calf Growth Performance Data Measurement of growth-related hormone levels in calves: The levels of insulin (INS), growth hormone (GH), somatostatin (SS), insulin-like growth factor 1 (IGF-1), insulin-like growth factor binding protein 2 (IGFBP-2), insulin-like growth factor binding protein 3 (IGFBP-3), glucagon-like peptide-2 (GLP-2), and growth hormone-releasing hormone (GHRH) in calf serum were detected according to the ELISA kit instructions (Jingmei Biotechnology Co., Ltd.). The required strips were removed from the aluminum foil bag after equilibration at room temperature for 20 min, and the remaining strips were sealed in a resealable bag and returned to 4℃. Standard wells and sample wells were prepared. 50 μL of different concentrations of standard solution were added to each standard well; 10 μL of the sample to be tested was added to each sample well, followed by 40 μL of sample diluent; no diluent was added to the blank wells. Except for the blank wells, 100 μL of horseradish peroxidase (HRP)-labeled detection antibody was added to each standard and sample well. The reaction wells were sealed with sealing film and incubated in a 37℃ water bath or incubator for 60 min. Discard the liquid, blot dry on absorbent paper, fill each well with washing buffer, let stand for 1 minute, shake off the washing buffer, blot dry on absorbent paper, and repeat this washing process 5 times (or a plate washer can be used). Add 50 μL each of substrate A and B to each well, and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well, and within 15 minutes, measure the OD value of each well at a wavelength of 450 nm.

[0027] like Figure 1 and Figure 2 As shown, throughout the entire experimental period, the GH levels of calves in the low RPG group were significantly higher than those in the high RPG group, and were also significantly higher than those in the control group at days 21 and 28 (P < 0.01). Figure 1 A). Calves in the low-RPG group had significantly higher GLP-2 levels at days 7, 21, and 28 than those in the high-RPG group (P < 0.01) and the control group (P < 0.05). Figure 1 (B) indicates that feeding RPG to periparturient yaks promotes the secretion of growth-related hormones in calves. Regarding GHRH, the GHRH levels in the low-RPG group were significantly higher than those in the high-RPG group throughout the entire experimental period, and were also significantly higher than the control group at days 21 and 28 (P < 0.05). Figure 1 C). Regarding IGF-1, the serum IGF-1 level in the low-RPG group was significantly higher than that in the high-RPG group throughout the entire experimental period (P < 0.05), and was significantly higher than that in the control group at 7 days and 14 days of low RPG (P < 0.01). Figure 1 D).

[0028] Regarding IGFBP-2 levels, the low-RPG group calves showed significantly higher IGFBP-2 levels at 7, 14, and 28 days than the control group (P < 0.01), and significantly higher levels at 28 days than the high-RPG group (P < 0.01). Figure 2A) Feeding calving mother yaks 150g RPG during the calving period can improve the growth performance and enhance the metabolic capacity of their calves. Regarding IGFBP-3, the serum IGFBP-3 level in the low-RPG group was significantly lower than that in the control group throughout the entire experimental period (P < 0.05), and significantly lower than that in the high-RPG group at days 14, 21, and 28 (P < 0.01). Figure 2 B). Regarding INS levels, the low-RPG group calves had significantly higher INS levels on days 7 and 14 than the high-RPG group (P < 0.05), and significantly higher INS levels on days 21 and 28 than the control group (P < 0.01). Conversely, the high-RPG group had significantly higher INS levels on days 21 and 28 than the control group (P < 0.05). Figure 2 C). Regarding SS levels, the levels in the low RPG group calves at 14, 21, and 28 days were significantly lower than those in the control group and the high RPG group (P < 0.01). Figure 2 D).

[0029] Determination of serum biochemical parameters in calves: The levels of glucose (GLU), β-hydroxybutyrate (BHBA), total cholesterol (TC), triglycerides (TG), albumin (ALB), total protein (TP), and alanine aminotransferase (ALT) in serum were measured using a Catalyst One blood biochemistry analyzer. After the sample had returned to room temperature, it was gently inverted ten times before being fed into the analyzer for testing.

[0030] like Figure 3 As shown, the serum BHBA concentration in the low RPG group was significantly lower than that in the high RPG group at 7, 14, and 28 days (P < 0.05), and the BHBA concentration was significantly lower than that in the control group throughout the entire experimental period (P < 0.05). Figure 3 A). The serum ALB concentration in the low RPG group was significantly higher than that in the high RPG group throughout the entire experimental period (P < 0.01), and significantly higher than that in the control group at days 21 and 27 (P < 0.01). Figure 3 B). Regarding TG, calves with low RPG levels had significantly lower levels than the control group at 14, 21, and 28 days (P < 0.05), and were significantly lower than the high RPG group at 14 and 28 days (P < 0.05). Meanwhile, calves in the high RPG group had significantly lower serum TG levels than the control group at 14 and 21 days (P < 0.05). Figure 3 C). Regarding ALT, the ALT levels in calves in the low RPG group were significantly lower than those in the high RPG group at 7, 14, and 28 days, and significantly lower than those in the control group at 7, 14, and 21 days (P < 0.01). Figure 3(D) This indicates that low RPG intake in calving cows had no significant negative impact on the hepatocytes of calves. Regarding TP content, calves in the low RPG group were significantly higher than those in the high RPG group throughout the entire experimental period (P < 0.01), and the TP level in the control group was also significantly higher than that in the high RPG group (P < 0.05). Figure 3 E). Regarding serum TC, the serum TC levels in calves of the low RPG group were significantly lower than those in the control group at 14, 21, and 28 days (P < 0.05), and the serum TC levels in calves of the high RPG group were also significantly lower than those in the control group at these three periods (P < 0.05). Figure 3 F). Serum GLU levels in the low-RPG group were significantly higher than those in the control group at days 14, 21, and 28 (P < 0.05). Furthermore, at day 14, the GLU level in the low-RPG group was significantly higher than that in the high-RPG group (P < 0.05), while the GLU level in the high-RPG group at day 28 was significantly higher than that in the control group (P < 0.05). Figure 3 G).

[0031] Determination of calf immune indicators: The levels of bovine immunoglobulin G (IgG), bovine interleukin 6 (IL-6), bovine interleukin 10 (IL-10), and bovine tumor necrosis factor alpha (TNF-α) in calf serum were detected according to the instructions of the ELISA kit (Jingmei Biotechnology Co., Ltd.).

[0032] Remove the plate strips from the aluminum foil bag after equilibration to room temperature for 20 minutes. Seal the remaining strips in a sealing bag and return to 4°C. Set up standard and sample wells. Add approximately 50 μL of different concentrations of standard to each standard well. Add 10 μL of the test sample to each sample well, followed by approximately 40 μL of sample diluent. Do not add any to the blank wells. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP)-labeled detection antibody to each standard and sample well. Seal the reaction wells with sealing film and incubate in an incubator or 37°C water bath for 60 minutes. Discard the liquid, pat dry on absorbent paper, then fill each well with washing buffer, let stand for 1 minute, discard the washing buffer, and pat dry on absorbent paper. Repeat this washing process 5 times (or use a plate washer). Add 50 μL of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 min.

[0033] like Figure 4As shown, throughout the entire experimental period, the TNF-α level in the low-RPG group was significantly lower than that in the control group (P < 0.05), and at days 14, 21, and 28, the TNF-α levels in calves of the low-RPG group were significantly lower than those in the high-RPG group (P < 0.01). Figure 4 B), and the serum IL-6 concentration in the low RPG group was significantly lower than that in the high RPG group at 14, 21, and 28 days (P < 0.01). Figure 4 C), and at 14 and 28 days, it was also significantly lower than the control group (P < 0.05). Regarding IL-10 and IgG concentrations ( Figure 4 D; Figure 4 (A) There were no significant differences in IgG levels among the three groups at any of the four time periods. However, calves born to cows fed 150 g RPG during the peripartum period showed significantly lower TNF-α and IL-6 concentrations, indicating that RPG can positively regulate the immune capacity of calves and ensure their healthy growth.

[0034] Determination of oxidative and antioxidant indices in calves: According to the kit instructions (Nanjing Jiancheng Bioengineering Institute), superoxide dismutase (SOD), catalase (CAT), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) in calf serum were detected. The separated serum was analyzed using an enzyme-linked immunosorbent assay (ELISA) with a microplate reader to determine the levels of SOD, CAT, MDA, and T-AOC, following the specific procedures outlined in the kit instructions.

[0035] The specific steps are as follows: Number the test tubes, add 0.1 mL of sample and 1 mL of reagent one, and preheat all test tubes in a 37℃ water bath for 3-5 minutes. During testing, add 0.1 mL of reagent two to each tube, mix immediately and time accurately, and place in a 37℃ water bath. After 60 seconds of reaction, immediately add reagent three (to terminate the reaction) and mix well. Test the subsequent test tubes sequentially, and finally add reagent four to all test tubes together, mix well, and read the value using a 405 nm wavelength microplate reader. Calculate serum CAT activity according to the formula in the instruction manual. Prepare the reagents according to the instruction manual, add serum sequentially to centrifuge tubes, cap the centrifuge tubes, poke a small hole in the cap with a needle, mix with a vortex mixer, and place in a 95℃ water bath (or boil in a pot with the lid off) for 40 minutes. After removal, cool under running water, and then centrifuge at 3500-4000 rpm for 10 minutes (if the speed is lower than 3000 rpm, the centrifugation time needs to be extended to ensure complete precipitation). Collect the supernatant, zero the microscope at 532 nm and 1 cm optical path using distilled water, and then measure the absorbance of each tube. Calculate the MDA content strictly according to the formula in the instructions. Add serum sequentially to centrifuge tubes, add the corresponding enzyme working solution, mix well, and incubate at 37°C for 20 minutes. Then, read the absorbance at 450 nm using a microplate reader and calculate the serum SOD content according to the formula in the instructions. Add serum sequentially to centrifuge tubes, add reagent four application solution and ABTS working solution sequentially according to the test instructions, react at room temperature for 6 minutes, and then read the absorbance at 405 nm using a microplate reader. Create a standard curve with the OD values ​​of the standards as the x-axis and the corresponding standard concentrations as the y-axis. Then, generate the curve formula using graphing software (or Excel). Substitute the OD values ​​measured in the test tubes into the calculation formula to obtain the T-AOC content in the serum. See the appendix for detailed reagent composition and preparation.

[0036] like Figure 5 As shown, the serum CAT concentration in calves in the low PRG group was significantly higher than that in the control group and the high PRG group throughout the entire experimental period (P < 0.05). Figure 5 A), and at day 28, the CAT level in calves of the high RPG group was significantly lower than that in the control group (P < 0.05). Regarding MDA, the serum MDA levels in calves of both the low RPG and control groups were significantly lower than those in the high RPG group throughout the entire experimental period (P < 0.01). Figure 5 B). Regarding SOD, the SOD levels of calves in the low PRG group and the control group were significantly higher on day 7 than those in the high PRG group (P < 0.05). Figure 5 C), and at day 28, the SOD level in calves of the low RPG group was significantly higher than that in the control and high RPG groups (P < 0.05). The serum T-AOC level in calves of the low RPG group was significantly higher at day 28 than that in the control and high RPG groups (P < 0.05). Figure 5 D).

[0037] Determination of microbial indicators in calf feces: All samples were stored on dry ice and sent to Shanghai Meiji Biotechnology Co., Ltd. for testing. DNA was extracted from the total genome of the microbial community according to the manufacturer's instructions, and then detected by 1% agarose gel electrophoresis. The concentration and purity of the extracted genomic DNA were determined using a NanoDrop2000. Using the extracted DNA as a template, PCR amplification of the full-length 16S rRNA gene or ITS gene was performed using barcode-enabled primers. After detection by 2% agarose gel electrophoresis, the DNA was purified by magnetic beads, and then the purified product was quantified using a Qubit 4.0 analyzer. Samples were mixed according to the required sequencing volume for each sample. Library construction was performed using the SMRTbell prep kit 3.0, including DNA damage repair, end repair, and adapter ligation. Sequencing was performed using the PacBio Sequel IIe System (Shanghai Meiji Biotechnology Co., Ltd.). HiFi reads generated from the sequenced subreads were used for subsequent data analysis using SMRT-Link v11.0 in CCS mode.

[0038] Using UPARSE 7.1, OTU clustering and chimera removal were performed on sequences based on 97% similarity. Chloroplast and mitochondrial sequences annotated in all samples were removed. To reduce the impact of sequencing depth on subsequent Alpha and Beta diversity data analysis, the sequence count of all samples was flattened to 6,000. After flattening, the average sequence coverage of each sample still reached 99.09%. OTU taxonomic annotation was performed using the RDP classifier against the Silva 16S rRNA gene database, with a confidence threshold of 70%. Community composition of each sample was analyzed at different taxonomic levels. 16S functional prediction analysis was performed using PICRUSt2 software.

[0039] Alpha diversity indices were calculated using Mothur software, and inter-group differences in alpha diversity were analyzed using the Wilcoxon rank-sum test. PCoA analysis based on the Bray-Curtis distance algorithm was used to examine the similarity between microbial community structures, and the significance of differences in microbial community structure between sample groups was determined using the PERMANOVA nonparametric test. LEfSe analysis (LDA>2, P<0.05) was used to identify bacterial groups with significant differences in abundance at the phylum to genus level among different groups. Distance-based redundancy analysis was applied to investigate the influence of soil physicochemical indicators on soil bacterial community structure. Linear regression analysis was used to evaluate the impact of the main soil physicochemical indicators identified in the db-RDA analysis on microbial alpha diversity. Species were selected based on Spearman correlation (|r|>0.6, p<0.05), and correlation network diagram analysis was performed.

[0040] like Figure 6 and Figure 7 As shown, the number of OTUs in the three experimental groups increased rapidly with the increase of sequencing data, and the Sobs dilution curves rose rapidly. Then, as the sequencing data gradually increased to a certain level, the rate of increase in the number of OTUs gradually slowed down, and the rise in the Sobs dilution curves tended to decrease. When the sequencing data reached 40,000 reads, the Sobs dilution curves of the three experimental groups finally stabilized. This indicates that the effective sequence quantity of each sample in the three groups was sufficient to cover most of the microbial species in the calf manure microbial community. This suggests that the sequencing depth at this point can represent the richness of species diversity in the overall samples of the three groups. The Shannon dilution curves show that when the sequencing data reached approximately 40,000 reads, the dilution curves of the three groups tended to flatten out. This indicates that the sequencing data at this point already included all the microbial communities in the manure samples. As the sequencing data continued to increase, the microbial diversity of the three groups no longer increased. A larger range of the curve on the horizontal axis indicates higher species richness; the flatness of the curve reflects the evenness of the community in the sample. The flat curve in the figure indicates a uniform distribution of species.

[0041] like Figure 6 and Figure 7 As shown, 28-day-old calves in different groups produced a total of 766 OTUs, of which 231 were common OTUs. The control group had 463 OTUs, including 126 unique OTUs. The low RPG group had 482 OTUs, including 137 unique OTUs. The high RPG group had 442 OTUs, including 113 unique OTUs.

[0042] Alpha diversity analysis primarily assesses the richness and diversity of microbial communities in environmental samples using multiple diversity indices, and explores differences in alpha diversity indices between control and treatment groups through inter-group difference tests. As shown in Table 3, commonly used metrics include chao, Shannon, ace, Simpson, and coverage. In yaks fed RPG, the Shannon index in the low-RPG group was significantly higher than that in the control group (P < 0.05), but there was no significant difference between the high-RPG group and the control group (P > 0.05). The ace index in the low-RPG group was significantly higher than that in the control and high-RPG groups (P < 0.01), but there was no significant difference between the control and high-RPG groups (P > 0.05). Regarding the Simpson index, the low-RPG group was significantly lower than that in the control group (P < 0.05), but there was no significant difference between the low-RPG group and the high-RPG group (P > 0.05). There were no significant differences in Ace, chao, coverage, and coverage among the groups (P > 0.05). The Shannon index of the low RPG group was significantly higher than that of the control group and the high RPG group, while the Simpson index was significantly lower than that of the control group and the high RPG group, which further confirms the increased community diversity in the low RPG group.

[0043] Table 3. Alpha diversity data of calf manure microorganisms Based on the taxonomic analysis results, the species composition at the phylum and genus levels of the experimental and control groups can be determined, as shown in Tables 4 and 5. Figure 9 A total of 16 major phyla were detected at the phylum level in both the control and experimental groups. The figure below shows the top 10 bacterial groups with the highest abundance at the phylum level in calf yak feces. Figure 9 A). The most abundant phyla in the gut microbiota of calves were Firmicutes, Bacteroidetes, and Proteobacteria. In the low RPG group, the proportions of these three phyla in the total gut microbiota were Firmicute (74.6%), Bacteroidetes (20.7%), and Proteobacteria (2.87%). In the high RPG group, the proportions were Firmicute (61.76%), Bacteroidetes (27.6%), and Proteobacteria (10.24%). In the control group, the proportions were Firmicute (46.33%), Bacteroidetes (36.4%), Proteobacteria (4.69%), and Fusobacteriota (12.51%). A total of 264 bacterial species (including unnamed classifications) were detected at the genus level in both the control and experimental groups. The figure below shows the top 10 most abundant bacterial groups at the genus level in calf feces. Figure 9In the control group, the most abundant gut microbiota genera were Bacteroides (33.86%), Lactobacillus (8.03%), and Fusobacterium (12.51%). In the low RPG group, the most abundant genera were Bacteroides (18.9%), Clostridia (9.02%), and Butyricicoccus (8.47%). In the high RPG group, the most abundant genera were Bacteroides (25.77%), Escherichia-Shigella (9.91%), and Lactobacillus (9.23%).

[0044] Table 4. Relative abundance analysis of bacteria at the phylum level (%) (Top 10 in relative abundance) Table 5. Relative abundance analysis of bacteria at the genus level (%) (Top 10 in relative abundance) Measurement of metabolomics parameters in calves: Serum samples were sent to Shanghai Meiji Biotechnology Co., Ltd. for testing. 100 μL of liquid sample was added to a 1.5 mL centrifuge tube, followed by 400 μL of extraction buffer. After vortexing for 30 seconds, the sample was extracted using low-temperature ultrasonication for 30 minutes. The sample was then placed at -20℃ for 30 minutes. The sample was centrifuged at 13000 g for 15 minutes at 4℃. The supernatant was collected, dried under nitrogen, and reconstituted with 100 µL of reconstitution solution. The sample was then extracted again using low-temperature ultrasonication for 5 minutes, followed by centrifugation at 13000 g for 10 minutes at 4℃. The supernatant was transferred to a vial with an internal insertion tube for analysis. The samples were analyzed by LC-MS / MS using a Thermo Fisher Scientific UHPLC-Q Exactive HF-X system. Raw LC-MS data were imported into the metabolomics processing software Progenesis QI for baseline filtering, peak identification, integration, retention time correction, and peak alignment, ultimately obtaining a data matrix containing retention time, mass-to-charge ratio, and peak intensity. Simultaneously, MS and MSMS mass spectrometry information were matched with the public metabolic database HMDB, as well as the Metlin and Meiji self-built libraries to obtain metabolite information. The data matrix after library searching was uploaded to the Meiji cloud platform for relevant analysis. Preprocessing of the data matrix included removing missing values ​​according to the 80% rule, retaining variables with more than 80% non-zero values ​​from at least one sample, and then filling in the missing values. To reduce errors caused by sample preparation and instrument instability, summation normalization was used to process the response intensity of sample mass spectrometry peaks, resulting in a normalized data matrix. Variables with a relative standard deviation (RSD) > 30% for QC samples were also removed, and logarithmic transformation was performed using log10 to obtain the data matrix used for subsequent analysis.

[0045] Principal component analysis and orthogonal least partial squares discriminant analysis were performed on the preprocessed data matrix using the ropls package in R. Seven-loop cross-validation was used to evaluate the model's stability. The selection of significantly different metabolites was based on the variable weights (VIP) obtained from the OPLS-DA model and the Student's t-test p-value; metabolites with VIP > 1 and p < 0.05 were considered significantly different metabolites.

[0046] Metabolic pathway annotation was performed on the differentially metabolites obtained using the KEGG database, revealing the pathways involved. Enrichment analysis of the metabolic pathways was conducted using the Python package scipy.stats, and Fisher's exact test was used to identify the biological pathways most relevant to the experimental treatment.

[0047] R²Y and Q² are used to evaluate the modeling and predictive capabilities of the PLS-DA model, respectively, as shown in Table 6. The larger the cumulative values ​​of R²Y and Q²Y, the more stable and reliable the model. If both R²Y and Q²Y are greater than 0.5, the model is accurate and reliable.

[0048] Table 6. Parameters of PLS-DA and OPLS-DA models The experiment was divided into three groups: a control group, a low-RPG group, and a high-RPG group. The metabolic set of the low-RPG group vs. control group contained 111 differentially expressed metabolites, the low-RPG group vs. high-RPG group contained 56 differentially expressed metabolites, and the high-RPG group vs. control group contained 50 differentially expressed metabolites. Two differentially expressed metabolites were shared across all three metabolic sets. The low-RPG group vs. control group and the high-RPG group vs. control group shared 9 identical differentially expressed metabolites, while the low-RPG group vs. high-RPG group shared 3 identical differentially expressed metabolites. The high-RPG group vs. control group and the high-RPG group vs. high-RPG group shared 4 identical differentially expressed metabolites. Figure 10 As shown in Table 7, in both positive and negative ion modes, 10 differentially expressed metabolites were upregulated and 22 were downregulated between the low RPG group and the control group in positive ion mode, and 20 differentially expressed metabolites were upregulated and 15 were downregulated between the low RPG group and the high RPG group. In the high RPG group and the control group, 11 differentially expressed metabolites were upregulated and 20 were downregulated in positive ion mode. In the low RPG group and the control group, 66 differentially expressed metabolites were upregulated and 13 were downregulated in negative ion mode, and 13 differentially expressed metabolites were upregulated and 6 were downregulated between the low RPG group and the high RPG group. In the high RPG group and the control group, 10 differentially expressed metabolites were upregulated and 9 were downregulated in negative ion mode.

[0049] Table 7. Number of Differential Metabolites under Positive and Negative Ions like Figure 11 , Figure 12 KEGG pathway enrichment analysis refers to the enrichment analysis of a selected metabolic set, using the hypergeometric distribution algorithm to identify pathways with significantly enriched metabolites within that set. In the metabolic sets of the low RPG group vs. the control group ( Figure 11 There are 19 metabolic pathways in total. Significantly differentially expressed metabolites are mainly enriched in α-linolenic acid metabolism, pyrimidine metabolism, purine metabolism, linoleic acid metabolism, biosynthesis of unsaturated fatty acids, and nucleotide metabolism. In the score plot, purine metabolism and nucleotide metabolism pathways are significantly downregulated, while linoleic acid metabolism, biosynthesis of unsaturated fatty acids, α-linolenic acid metabolism, pyrimidine metabolism, and PPAR pathways are significantly upregulated. Figure 12A); In the low RPG group vs. high RPG group, there were 7 metabolic pathways, and the significantly different metabolites were mainly enriched in primary bile acid biosynthesis, cholesterol metabolism, and ABC protein transport (A); Figure 11 Furthermore, cholesterol metabolism and primary bile acid biosynthesis were significantly downregulated in the score plot, while ABC protein transport was significantly upregulated. Figure 12 B); metabolic concentration in the high RPG group vs. the control group ( Figure 11 There are 39 metabolic pathways in total. Significantly differentially expressed metabolites are mainly enriched in autophagy-other, glycosylphosphatidylinositol (GPI)-anchored biosynthesis, autophagy-animal, ascorbic acid and aldehyde metabolism, Kaposi's sarcoma-associated herpesvirus infection, gap junctions, folic acid biosynthesis, synaptic vesicle circulation, unsaturated fatty acid biosynthesis, tryptophan metabolism, purine metabolism, nucleotide metabolism, and ABC protein transport. In the score graph, folic acid biosynthesis, unsaturated fatty acid biosynthesis, and ABC protein transport are significantly upregulated. Figure 12 C), while autophagy-others, glycosylphosphatidylinositol (GPI)-anchored biosynthesis, autophagy-animals, ascorbic acid and aldehyde metabolism, Kaposi's sarcoma-associated herpesvirus infection, gap junctions, synaptic vesicle circulation, tryptophan metabolism, purine metabolism, and nucleotide metabolism were significantly downregulated.

[0050] As shown in Table 8, among the differentially expressed metabolites between the low RPG group and the control group, oleic acid, cytosine, 13,16-docosahexaenoic acid, eicosapentaenoic acid, stearatetraenoic acid, 5-hydroxyeicosapentaenoic acid, and 5-hydroxy-6,8,11,14-eicosapentaenoic acid were significantly upregulated (P < 0.05), while leaf alcohol and other differentially expressed metabolites were significantly downregulated (P < 0.05).

[0051] Table 8. Differential metabolites between the low RPG group and the control group As shown in Table 9, among the differentially expressed metabolites between the high RPG group and the control group, guanosine, 6-hydroxy-2-aminopurine, vitamin C, 2,6-dihydroxypurine, indoleacetic acid, and 5-hydroxytryptamine were significantly downregulated (P < 0.05), while 7,8-dihydrobiopterin, BH2, 2'-deoxycytidine, 5-aminoketopentetrine, and L-valine were significantly upregulated (P < 0.05).

[0052] Table 9. Differential metabolites between the high RPG group and the control group Among the differentially expressed metabolites between the low-RPG and high-RPG groups, glycine chenodeoxycholic acid and 2'-deoxycytidine were significantly downregulated (P < 0.05), while pyruvicin and neomycin sulfate EP impurity A were significantly upregulated (P < 0.05). See Table 12 for details.

[0053] Table 10. Differential metabolites between the low RPG group and the high RPG group The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for supplementing yak hay with rumen-treated glucose during the dry season in alpine grasslands, characterized in that, Includes the following steps: S1. Select peripartum plateau yaks; S2. Implement phased feeding management for plateau yaks during the peripartum period: a. Feeding should begin 14 days before the periparturient period, including a 7-day pre-feeding period and a 42-day regular feeding period. The pre-feeding period is used to establish the feeding rhythm, and the yak's nutritional and metabolic indicators are continuously monitored during the regular feeding period. b. Daily feeding in different time periods: During the period from 9:00 to 10:30 in the morning, feed mixed hay containing rumen glucose in the pen; during the period from 17:00 to 18:30 in the afternoon, feed hay without rumen glucose in the pen alone. c. After feeding in the pen, drive the yaks to the surrounding plain area for free grazing and drinking.

2. The method for supplementing yak hay with rumen-treated glucose during the dry season in alpine grasslands according to claim 1, characterized in that, The specific method for preparing hay containing rumen glucose is as follows: Place 150g of rumen glucose and 300g of hay in a special feeding container, and add 50ml of water and stir to make the glucose evenly adhere to the surface of the hay.

3. The method for supplementing yak hay with rumen-treated glucose during the dry season in alpine grasslands according to claim 1, characterized in that, Rumen glucose is encapsulated by hydrogenated fats.

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