Use of a kind of graptophyllum for preparing additive for reducing methane emission of fattening beef cattle

Abstract

The present application relates to the field of livestock technology, and particularly relates to application of an additive for reducing methane emission of fattening beef cattle prepared from Gracilaria lemaneiformis. The Gracilaria lemaneiformis is natural sun-dried or oven-dried Gracilaria lemaneiformis. The present application adds the oven-dried Gracilaria lemaneiformis powder into the mixed basic daily ration, finds that it can inhibit the activity of the methanogenic archaea, increase the abundance of the bacterial genus competing with the methanogenic archaea for the substrate, and optimize the fermentation process by adjusting the relative proportion of volatile fatty acids such as acetic acid, propionic acid and butyric acid in the rumen, thereby significantly reducing the methane emission of ruminants, having important value and significance in green emission reduction of the breeding industry, and having great industrial application potential.

Description

TECHNICAL FIELD

[0001] The application relates to the field of livestock farming, and particularly relates to application of an additive prepared from Gracilaria lemaneiformis for reducing methane emission of fattening beef cattle. BACKGROUND

[0002] According to the report of the Food and Agriculture Organization of the United Nations (FAO), more than one-third of global greenhouse gas emissions are derived from the agricultural and food system, in which methane (CH4) produced by rumen microorganisms of ruminants is the main emission source, accounting for about 18% of the total global greenhouse gas emissions. In the greenhouse gas emissions of the livestock industry, the emissions of beef cattle and dairy cattle account for 35% and 30% of the total amount respectively, followed by sheep and goats. The data of the FAO and the United States Department of Agriculture (USDA) show that, as of 2022, the global beef cattle inventory reached 944 million. China is the largest ruminant breeding country in the world, with more than 100 million beef cattle. Therefore, developing a feed additive that is efficient, low-cost and green and sustainable to reduce methane emission of fattening beef cattle is an urgent problem to be solved in the livestock industry.

[0003] The nutrients in the diet of ruminants are subjected to anaerobic fermentation under the action of various rumen microorganisms, are decomposed and generate microbial protein (MCP), volatile fatty acid (VFA), hydrogen (H2) and carbon dioxide (CO2) and the like. These substances provide protein and energy for the animal body, but at the same time also provide substrates such as H2, CO2, formate, acetate and methyl compounds for the generation of methane.

[0004] The methane generation mechanism involves a complex microbial metabolic network, and is regulated by environmental conditions such as animal diet, pH value of rumen, temperature and the like. Among them, the composition of feed is one of the key factors affecting methane generation. Domestic and foreign researches show that adding an appropriate amount of large seaweed in the feed of ruminants can reduce the methane emission of ruminants, such as Codium fragile and Sargassum muticum. However, the high concentration of halogenated compounds contained in Codium fragile is not conducive to the health of animals, and the methane emission reduction effect of Sargassum muticum is general. Therefore, it is particularly important to find a feed additive that can effectively reduce the methane emission of ruminants and does not have negative effects on the health of animals. SUMMARY

[0005] To solve the above problems, the application discloses application of an additive prepared from Gracilaria lemaneiformis for reducing methane emission of fattening beef cattle. The additive is added to the feed of fattening beef cattle, can effectively regulate the microbial composition of rumen, reduce the synthesis of methane, achieve the effect of emission reduction, and does not have negative effects on the health of animals.

[0006] Meanwhile, the application further provides a feed for reducing methane emission of Simmental crossbred cattle.

[0007] To achieve the above object, the technical scheme adopted by the present application is:

[0008] An application of using Gracilaria lemaneiformis to prepare an additive for reducing methane emission of fattening beef cattle.

[0009] Gracilaria lemaneiformis of the Gracilaria genus of the Gracilaria family is a kind of natural seaweed resource, which contains rich nutritional ingredients such as polysaccharides, proteins, minerals and vitamins, and does not contain halogenated compounds harmful to animals. Through specific processing, Gracilaria lemaneiformis can be prepared into an additive suitable for being added to the feed of fattening beef cattle. The additive can inhibit the activity of methanogenic archaea and increase the abundance of bacteria genera competing with methanogenic archaea for substrates, thereby reducing the synthesis of CH4.

[0010] As a preferred embodiment of the feed for reducing methane emission of fattening beef cattle according to the present application, the addition amount of the algal powder of Gracilaria lemaneiformis is 2wt%-10wt% of the dry matter of the basic daily ration of the fattening beef cattle; preferably, the addition amount of the algal powder of Gracilaria lemaneiformis is 2wt%-5wt% of the dry matter of the basic daily ration of the fattening beef cattle.

[0011] As a preferred embodiment of the feed additive according to the present application, the Gracilaria lemaneiformis can be any Gracilaria lemaneiformis purchased through commercial channels or isolated by oneself.

[0012] As a preferred embodiment of the feed additive according to the present application, the drying process of Gracilaria lemaneiformis is one of natural airing or drying.

[0013] Meanwhile, the present application also discloses a feed for reducing methane emission of fattening beef cattle, which contains 2wt-10wt% of Gracilaria lemaneiformis in dry matter.

[0014] In the above-mentioned feed, the Gracilaria lemaneiformis is algal powder obtained by drying and crushing Gracilaria lemaneiformis.

[0015] In the above-mentioned feed, the addition amount of Gracilaria lemaneiformis is 2wt%, and the methane emission reduction rate of Simmental crossbred cattle during the fattening period reaches 11.4%-18.4%.

[0016] The application takes 0.3 g of corn stalks and 0.2 g of concentrate (60:40 of roughage: concentrate) as a mixed basic daily ration, and adds the Gracilaria dura powder into the daily ration according to 2%, 5%, 10% of the dry weight of the mixed basic daily ration. Through the in vitro rumen fermentation test, it is found that the Gracilaria dura powder can reduce the proportion of methanogens in the rumen, and reduce the methane emission of the bovine rumen by 34.4%. In addition, it is also found that the addition of the Gracilaria dura powder can also reduce the content of NH3-N, acetic acid and butyric acid, and increase the content of propionic acid. The application determines that the addition of the Gracilaria dura in the mixed basic daily ration of the fattening beef cattle can reduce the methane emission of the fattening beef cattle, and has important application prospects for the green emission reduction of the breeding industry.

[0017] The present study first found that the Gracilaria dura as a feed additive for fattening beef cattle can regulate the relative proportion of volatile fatty acids such as acetic acid, propionic acid and butyric acid in the rumen, optimize the fermentation process, promote the proliferation of beneficial bacteria and effectively inhibit the activity of methanogens. In addition, the Gracilaria dura can also induce the synthesis of unsaturated fatty acids such as 13(S)-HOTrE and 9(S)-HOTrE, and then target the metabolic pathway of methanogenic archaea, further inhibiting its activity. Overall, the Gracilaria dura reduces methane emission through multiple pathways such as regulating rumen VFA spectrum, remodeling microbial community structure and inducing synthesis of specific metabolites.

[0018] The results of the fattening beef cattle animal experiment show that when the Gracilaria dura is not added, there is no significant difference in CH4 emission between each group. On the 15th day and the 30th day of the experiment, the CH4 emission of the group with 2% Gracilaria dura added in the feed shows a downward trend (0.05≤P<0.10), which is reduced by 18.4% and 12.4% respectively compared with the control group. By the 60th day, the CH4 emission of the 2% addition group is significantly reduced by 11.4%.

[0019] Overall, the Gracilaria dura reduces methane emission through multiple pathways such as regulating rumen VFA spectrum, remodeling microbial community structure and inducing synthesis of specific metabolites. The animal experiment results show that the addition of 2% Gracilaria dura can significantly reduce the methane emission in the rumen of beef cattle, and has no toxicity risk, which provides a scientific basis for the application of Gracilaria dura in beef cattle large-scale breeding.

[0020] The beneficial effects of the application are:

[0021] 1. The Gracilaria dura added to the feed of fattening beef cattle can effectively regulate the microbial composition in the rumen, reduce the synthesis of methane, and achieve the effect of emission reduction;

[0022] 2. The Gracilaria dura regulates the relative proportion of volatile fatty acids such as acetic acid, propionic acid and butyric acid in the rumen, optimizes the fermentation process, promotes the proliferation of beneficial bacteria and effectively inhibits the activity of methanogens;

[0023] 3. Gracilaria lemaneiformis can also induce the synthesis of unsaturated fatty acids such as 13(S)-HOTrE and 9(S)-HOTrE, and further target to block the metabolic pathways of methanogenic archaea, thereby further inhibiting their activity;

[0024] Overall, Gracilaria lemaneiformis can reduce methane emissions by regulating rumen VFA spectrum, remodeling microbial community structure, and inducing synthesis of specific metabolites, etc. Animal experiment results show that the addition of 2% Gracilaria lemaneiformis can significantly reduce methane emissions in the rumen of beef cattle, and has no toxicity risk, which provides a scientific basis for the application of Gracilaria lemaneiformis in beef cattle large-scale breeding. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a schematic diagram of methane test time arrangement of Simmental crossbred cattle in the in vivo experiment of the present application;

[0026] Figure 2 is a methane correction curve in the in vivo experiment of the present application;

[0027] Figure 3 is a comparison chart of the relative abundance at the genus level of rumen bacteria (A) and methanogenic archaea (B) of the CON group (without the addition of seaweed) and the DG2 group (the addition amount of Gracilaria lemaneiformis is 2wt%) in the in vivo experiment of the present application. DETAILED DESCRIPTION

[0028] For the purpose of better illustrating the present application, technical solutions and advantages, the present application will be further described in combination with specific experiments.

[0029] In vitro experiment

[0030] Asparagopsis taxiformis, Scagassum maclurei and Gracilaria lemaneiformis samples were collected from Naozhou Island (North Latitude 20°56', East Longitude 110°33') in Guangdong Province, Weizhou Island (North Latitude 21°03', East Longitude 109°07') in Guangxi Zhuang Autonomous Region and Dongshan Island (North Latitude 23°43', East Longitude 117°25') in Zhangzhou City, Fujian Province, China.

[0031] After collection, all seaweed samples were first rinsed thoroughly with clean water to remove surface impurities and contaminants. After cleaning, the samples were dehydrated and divided into two groups, each using a different drying method. One group of samples was oven-dried at 65°C for 48 h to constant weight, while the other group used freeze-drying technology to maximize the retention of active ingredients and nutrients in the seaweed. After drying, all samples were ground into fine powder using a grinder and sieved through a 40-mesh screen to ensure uniformity and consistent particle size. The treated seaweed powder was stored at -20°C for subsequent analysis and use.

[0032] Six replicates were set up, with 0.3 g of corn straw and 0.2 g of concentrate (60:40 of roughage to concentrate) added to each fermentation bottle as the basic fermentation substrate. The control group (CON) did not add any seaweed treatment; the experimental groups added freeze-dried or oven-dried seaweed samples at 2%, 5%, and 10% of dry matter basis (DM), respectively. The nutritional composition of the basic fermentation substrate and the macroalgae used in the test is shown in Table 1, and the mineral content of the three macroalgae is shown in Table 2. To ensure the reliability of the results, the in vitro fermentation test of the three seaweeds was carried out in batches, with two replicates per batch.

[0033] On the day of the test, the rumen fluid of three Holstein cows was collected 2 hours after morning feeding (the test was conducted over three days). The collected rumen fluid was filtered through four layers of gauze and mixed, and then quickly transferred to a preheated thermos bottle that had been pre-flushed with carbon dioxide to maintain the activity of the rumen fluid. After collection, the rumen fluid was immediately transported to the laboratory. Upon arrival at the laboratory, the rumen fluid was mixed with buffer at a volume ratio of 1:2 at 39°C to form the fermentation liquid. To maintain an anaerobic environment, carbon dioxide was continuously introduced during the mixing process. The buffer was prepared according to the method of Menke et al. (Menke et al., 1979). Subsequently, the fermentation liquid was divided into 75 mL volumes and placed in fermentation bottles. The fermentation bottles were sealed with butyl rubber stoppers and aluminum caps and placed in a water bath shaker for fermentation at 39°C and 85 r / min for 48 h.

[0034] Table 1 Nutritional composition of substrates and macroalgae for in vitro rumen fermentation test (dry matter basis)

[0035] Item Corn stover Concentrate S. mooreana A. taxiformis G. lemaneiformis DM (dry matter basis) 26.26 93.53 NA NA NA OM (organic matter) 92.85 92.81 65.63 63.17 65.60 CP (crude protein) 7.12 20.47 12.72 22.61 25.00 NDF (neutral detergent fiber) 38.49 16.61 20.86 38.39 14.59 ADF (acid detergent fiber) 21.69 6.22 16.89 13.23 5.53 Ash (ash) 7.14 7.18 34.37 36.83 34.40

[0036] Table 2 Mineral content of three macroalgae

[0037] Item (mg / kg) G. lemaneiformis S. mooreana A. taxiformis Macro-minerls (macro-nutrients) Ca 2245.8 14045.7 49838.2 K 13721 76250.2 16664.6 Mg 2674 6431.6 8864.3 Na 11651 15780 19911 Trace elements (micro-nutrients) Al 2664.4 975.4 10944.1 B 177.8 156.3 75.3 Co 0.4971 0.4734 2.992 Cr 2.65 4.13 118.80 Cu 6.03 2.31 7.81 Fe 1204 628.7 7060.5 Mn 135.50 78.56 211.12 Mo 0.24 0.24 0.87 Sb 0.3802 0.2460 0.7309 Se 0.4849 0.4846 1.6183 Zn 35.56 9.60 36.46 Toxic heavy metals (toxic metals) As 6.03 102.94 10.04 Ba 6.39 20.66 34.97 Cd 0.60 1.94 0.50 Hg 0.0022 0.0030 0.0029 TI 0.0024 0.0018 0.1303 Pb 2.41 1.58 4.96

[0038] In vivo experiment

[0039] Eighteen Simmental crossbred beef cattle with similar age and weight of 400 kg were selected, and the basic diet was composed of Table 3, and the basic diet nutrient level was Table 4. The cattle were fed twice a day at 9:00 and 15:00, and free access to feed and water.

[0040] The animal experiment was carried out in Guangzhou Baiyun District Guangcongjiulu No. 1 Herbivore Experimental Base from November 4, 2024 to January 10, 2025, including a 14-day pre-test period and a 60-day test period.

[0041] Table 3 Basic diet group of Simmental crossbred beef cattle (dry matter basis)

[0042] Raw materials Content (%) Corn 43.4 Bran 5.0 Soybean meal 10.0 Wheat straw 30.0 Whole plant silage corn 10.0 NaCl 0.5 calcium carbonate CaCO3 0.6 Premix 0.5 Total 100

[0043] Table 4 Nutrient level of basic diet of Simmental crossbred beef cattle (dry matter basis)

[0044] Nutrient levels Content (%) Crude protein 12.05 Neutral detergent fiber 30.94 Acid detergent fiber 19.01 Crude fat 4.81 Calcium 0.76 Phosphorus 0.33 Net energy metabolizable (MJ / kg) 6.38

[0045] Before methane determination, the fattening beef cattle (Simmental crossbred beef cattle) underwent a 30-day diet adaptation period to ensure the stability of the rumen microbial flora. After the adaptation period, the GreenFeed system was used to determine the CH4 emission of each cattle (14d). Based on the above methane emission data, 2% and 5% of the dry powder of Gracilaria lemaneiformis were added to the basic diet, and then a 60-day feeding test was formally carried out. The first week of the test was the adaptation period of Gracilaria lemaneiformis, during which no methane emission determination was carried out. From the second week, the CH4 emission was determined for three consecutive days every week. The specific test arrangement is shown in Figure 1 At the same time, the feed intake of beef cattle was recorded, and the feed samples were placed in a 65℃ oven for 48 h to determine the dry matter content.

[0046] Performance test

[0047] In vitro experiment related test

[0048] (1) Gas sample collection and index determination

[0049] Gas samples produced by fermentation were collected using a syringe at 2, 4, 8, 12, 24 and 48 h after fermentation, and stored in single-valve aluminum foil gas collection bags for subsequent gas component analysis.

[0050] CH4 and CO2 concentrations were determined using a gas chromatograph (SP-2060T, Beijing Beifen-Tianguan), which was equipped with a 5A stainless steel chromatographic column (Φ3 mm x 3 m, support 60-80 mesh Chromosorb) and a Tbx-01 stainless steel chromatographic column (Φ3 mm x 1 m, support 60-80 mesh Chromosorb). The sample injection volume was set to 1 mL, the carrier gas was high-purity argon (content >= 99.999%), and the flow rate was 30 mL / min. The gas pressure was adjusted to 0.5 MPa. The temperatures of the detector (TCD) and the chromatographic column were both set to 100°C. The relevant test results are shown in Tables 5, 6, and 7, where CON represents no addition of seaweed, a,b,c P<0.05. The active ingredients in Asparagopsis taxiformis and Gracilaria lemaneiformis were also tested, and the test results are shown in Table 8.

[0051] Table 5 Effect of Asparagopsis taxiformis with different treatments and doses on gas production parameters of in vitro rumen fermentation

[0052]

[0053] Table 6 Effect of Gracilaria lemaneiformis with different treatments and doses on gas production parameters of in vitro rumen fermentation

[0054]

[0055] Table 7 Effect of Sargassum muticum with different treatments and doses on gas production parameters of in vitro rumen fermentation

[0056]

[0057] Table 8 Active ingredients (top 10) in Asparagopsis taxiformis and Gracilaria lemaneiformis

[0058]

[0059] Table are toxic and harmful substances.

[0060] (2) Rumen fluid sample collection and index determination

[0061] Ruminal fermentation parameters were mainly determined by measuring VFA and NH3-N concentrations in the fermentation broth. Immediately after the end of the experiment, the fermentation bottles were placed in ice water to terminate fermentation. The contents of the fermentation bottles were filtered with 300-mesh nylon bags, and then dried at 65°C for 24 h to a constant weight. 2 mL of fermentation broth was collected for VFA determination; 1 mL for NH3-N concentration analysis; 5 mL for 16s rRNA and metabolomics determination. All samples were stored at -80°C for subsequent analysis. NH3-N was determined using the method of Choi et al. (Choi et al., 2022). VFA was determined by gas chromatography (Agilent 6890N) (Wischer et al., 2013). See Table 9 for the results of the relevant tests.

[0062] Table 9. Effect of different processing methods and additive doses of Gracilaria on rumen fermentation parameters

[0063]

[0064] CON: no seaweed added, a,b,c P < 0.05.

[0065] (3) Rumen fluid 16s rRNA and metabolomics

[0066] Total microbial genomic DNA was extracted from rumen fluid samples using MP-soil (MP Biomedicals, USA). The quality and concentration of extracted DNA were determined by 1.0% agarose gel electrophoresis and NanoDrop 2000 spectrophotometer, and stored at -80℃. Bacterial primers were 338F_806R (Liu et al., 2016) and methanogenic archaea primers were 349F_806R (Wei et al., 2019) for PCR amplification. The PCR reaction system contained 2x Pro Taq (10 μL), forward and reverse primers (5 μM each, 0.8 μL), and template DNA (10 ng / μL). The amplification conditions were as follows: 95℃ pre-denaturation for 3 min, followed by 29 cycles of 95℃ denaturation for 30 s, 53℃ annealing for 30 s, and 72℃ extension for 45 s. The amplification products were detected by 2% agarose gel electrophoresis and purified using a DNA gel recovery purification kit (PCR Clean-Up Kit, Yewhua). The purified products were quantified using Qubit 4.0 (ThermoFisher Scientific, USA) and mixed in proportion according to the sequencing requirements. The library construction used the NEXTFLEX Rapid DNA-Seq Kit (Bioo Scientific, USA), and the steps included: (1) linker ligation; (2) magnetic bead screening to remove self-ligation fragments; (3) PCR amplification to enrich library templates; (4) magnetic bead recovery of PCR products to obtain the final library. The final library was sequenced by the Illumina Miseq PE300 or NovaSeq PE300 platform.

[0067] Take 100 μL of rumen fluid sample, add 400 μL of extraction solution (acetonitrile:methanol=1:1, containing 0.02 mg / mL L-2-chlorophenylalanine internal standard), vortex for 30 s, ultrasonic extraction at 5°C for 30 min (40 KHz), then stand at -20°C for 30 min, centrifuge at 4°C at 13000 g for 15 min, take the supernatant and nitrogen blow to dry, re-dissolve with 100 μL of re-dissolving solution (acetonitrile:water=1:1), ultrasonic extraction at 5°C for 5 min (40 KHz), centrifuge again at 4°C at 13000 g for 10 min, take the supernatant and transfer to a sample vial for testing. Mix all samples in equal volume to prepare quality control samples (QCs), and insert QC samples between every 5-15 samples. Use Thermo UHPLC-Q Exactive HF-X system for LC-MS / MS analysis. Chromatographic separation uses HSS T3 column (100 mm x 2.1 mm, 1.8 μm), mobile phase A is 95% water + 5% acetonitrile (containing 0.1% formic acid), mobile phase B is 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Positive ion mode gradient: 0-3 min, B phase from 0% to 20%; 3-4.5 min, to 35%; 4.5-5 min, to 100%; 5-6.3 min, maintain 100%; 6.3-6.4 min, to 0%; 6.4-8 min, maintain 0%. Negative ion mode gradient: 0-1.5 min, B phase from 0% to 5%; 1.5-2 min, to 10%; 2-4.5 min, to 30%; 4.5-5 min, to 100%; 5-6.3 min, maintain 100%; 6.3-6.4 min, to 0%; 6.4-8 min, maintain 0%. Flow rate 0.40 mL / min, column temperature 40°C. Mass spectrometry acquisition uses positive and negative ion modes, scan range 70-1050 m / z, sheath gas flow rate 50 psi, auxiliary gas flow rate 13 psi, heating temperature 425°C, positive and negative ion spray voltage 3500 V and -3500 V respectively, ion transmission tube temperature 325°C, collision energy 20-40-60 V cycle. First-order resolution 60000, second-order resolution 7500, data acquisition uses DDA mode. Relevant test results are shown in Figure 2 .

[0068] In vivo experiment related tests

[0069] The GreenFeed system needs to be calibrated for CH4 and CO2 gas before the test to ensure the accuracy and reliability of the measurement data. Calibrate the GreenFeed system for gas according to the method of Liu Zhuofan (Liu Zhuofan, 2023). The CH4 calibration results are as follows Figure 3, the methane release amount coincided with the baseline, meaning that the methane calibration was successful. The weights of the carbon dioxide gas cylinders before and after calibration were input into the GreenFeed website, and the CO2 recovery rates were calculated to be 95.46%, 97.82% and 101.7%, respectively, which were good recovery rates. The CH4 and CO2 calibration results were normal, and subsequent measurements could be performed.

[0070] The CH4 and CO2 contents were measured by a nondispersive infrared analyzer in the GreenFeed system. The formula was as follows (Hristov et al., 2015):

[0071] CH4volume = FC* CR * Σtp [Δt * (CH4avg-CH4 bkgrnd) * Qair]

[0072] wherein FC is a size conversion coefficient; CR is the capture rate of the gas (%); Δt is the time period for measuring the emission amount (s); CH4avg is the average concentration of CH4 during the measurement (%); CH4 bkgrnd is the environmental concentration of methane (%); and Qair is the airflow rate during the measurement (flow rate per unit time). After the volume of CH4 was calculated, the number of CH4 molecules per unit time could be calculated, and thus the mass concentration thereof was obtained. The calculation method for CO2 was the same as that for CH4. The relevant test results are shown in Table 10.

[0073] Analysis of results

[0074] Analysis of in vitro experimental test results:

[0075] From the test results of Table 5, Table 6 and Table 7, it can be observed that all three macroalgae can significantly reduce the CH4 emission in vitro. The freeze-dried Asparagopsis taxiformis has a significant effect on inhibiting CH4 emission (Table 5), with 5% and 10% addition reducing CH4 emission by 98.6% and 99.3%, respectively, which is better than the oven-dried treatment (82.7%). The CH4 emission reduction effect of Gracilaria lemaneiformis is affected by the additive amount, and reaches the emission reduction threshold at 5%. Among them, the oven-dried Gracilaria lemaneiformis at 2% and 5% reduces CH4 emission by 27.5% and 32.6%, respectively (Table 6). The treatment method and additive amount of Sargassum moserii have an interactive effect, and the addition of 2% freeze-dried Sargassum moserii can reduce CH4 emission by 18.8% (Table 7). All three macroalgae have the ability to alleviate rumen methane emission. Asparagopsis taxiformis can alleviate 37.8%-99.3% of rumen CH4 emission, Gracilaria lemaneiformis can reduce 27.5%-34.0%, and Sargassum moserii can reduce CH4 emission by 13.7%-24.2%. In addition, the differences in the CON control groups in Table 5, Table 6 and Table 7 are caused by different rumen fluid collection dates or individual rumen fluid differences. Among the three macroalgae, Asparagopsis taxiformis has the best methane emission reduction effect, but the high concentration of halogenated compounds contained therein is harmful to animal health (Table 8); and the emission reduction effect of Sargassum moserii is inferior to that of Gracilaria lemaneiformis.

[0076] From Table 9, it can be observed that the addition of Gracilaria lemaneiformis in feed reduces the concentration of NH3-N. NH3-N is the main raw material for the synthesis of microbial cell protein (MCP) in the rumen. About 50%-80% of small intestine can absorb protein from MCP, and its synthesis efficiency directly affects the protein utilization efficiency of ruminants. This indicates that rumen microorganisms inhibit protein hydrolysis and amino acid deamination or improve the utilization rate of NH3-N. The reduction of NH3-N production in the rumen shows that the addition of Gracilaria lemaneiformis in feed not only helps to improve the nitrogen utilization efficiency of rumen microorganisms, but also reduces the nitrogen emission of ruminants, which is beneficial to the environment.

[0077] In addition, from Table 9, it can be clearly observed that after adding Gracilaria lemaneiformis, the content of volatile fatty acids such as acetic acid and butyric acid is significantly reduced, while the content of propionic acid is relatively increased. Acetic acid is produced in the rumen, which releases H2, and H2 can be used as a hydrogen donor for methanogenic archaea to promote CH4 generation. In contrast, propionic acid acts as a sink for H2, competing with CH4 for metabolic H2. Therefore, the concentration of propionic acid in the rumen is negatively correlated with CH4 emission, while the concentration of acetic acid and the acetic acid to propionic acid ratio are positively correlated with CH4 emission. In this study, all experimental treatments significantly reduced the acetic acid to propionic acid ratio, indicating that part of H2 may be redistributed to the propionic acid production pathway, thereby reducing the amount of H2 available for CH4 generation, and achieving the effect of CH4 emission reduction.

[0078] From the microbial composition, the addition of Gracilaria can reduce the abundance of methanogenesis-related archaea, while increasing the abundance of genera that compete with methanogenic archaea and the genus Succinivibrio Figure 3 ). This change in microbial community further explains the mechanism of CH4 emission reduction. Metabolomics analysis showed that 131 differential metabolites were identified in rumen fluid, mainly involving carbohydrate metabolism, lipid metabolism, digestive system, and amino acid metabolism pathways. Notably, after the addition of Gracilaria, unsaturated fatty acids such as 13(S)-HOTrE and 9(S)-HOTrE, which inhibit the activity of methanogenic archaea, were up-regulated. This finding provides a new mechanism for the reduction of CH4 emission by Gracilaria.

[0079] In summary, this study confirms that Gracilaria can regulate the composition of volatile fatty acids (VFA) in the rumen, particularly by changing the relative proportions of acetic acid, propionic acid, and butyric acid, optimizing the fermentation process. Additionally, it can restructure the rumen microbial community, promoting the proliferation of beneficial bacteria while effectively inhibiting the activity of methanogenic bacteria. More importantly, Gracilaria can induce the synthesis of unsaturated fatty acids such as 13(S)-HOTrE and 9(S)-HOTrE, targeting the metabolic pathways of methanogenic archaea and inhibiting their activity. Overall, Gracilaria achieves the emission reduction goal through the synergistic effects of microbial community restructuring and metabolic network remodeling, providing a scientific basis for the development of new additives to reduce methane emissions from ruminants.

[0080] In vivo experimental test result analysis:

[0081] As shown in Table 10, the GreenFeed system monitoring results showed that there was no significant difference in CH4 emission between the groups without the addition of Gracilaria, and subsequent experiments could be conducted. On the 15th and 30th days of the experiment, the CH4 emission of the group with 2% Gracilaria added to the diet showed a downward trend (0.05 ≤ P < 0.10), with a decrease of 18.4% and 12.4%, respectively, compared to the control group. By the 60th day, the CH4 emission of the 2% addition group was significantly reduced by 11.4%. However, when the addition ratio increased to 5%, there was no significant difference in the effect on CH4 emission. This is inconsistent with the results of in vitro research, as the addition of 5% Gracilaria resulted in a more noticeable sea smell in the feed, reducing the feed intake of beef cattle and thus affecting the rumen fermentation process. In addition, the addition of 2% Gracilaria significantly reduced the CO2 emission of Simmental crossbred cattle, while the 5% Gracilaria group showed no significant change. Specifically, on the 15th, 30th, and 60th days of the experiment, the CO2 emission of the 2% Gracilaria group decreased by 20.0%, 16.2%, and 13.7%, respectively. The reduction trend of CO2 emission was consistent with the change in CH4 emission, both reaching a peak in the early stage and stabilizing in the middle and late stages.

[0082] Table 10 Effects of adding different levels of Gracilaria on CH4 and CO2 emissions of Simmental crossbred cattle

[0083] Time CON 2%wt 5%wt P CH4 / DMI (g / kg) Before experiment 28.89±4.11 27.63±2.30 30.38±3.07 0.365 15 d 27.63±3.17 22.52±1.99 25.92±4.91 0.070 30 d 28.06±1.78 24.58±2.18 28.08±3.85 0.069 60 d 29.94±1.43a 26.53±2.36b 29.40±1.61a 0.013 CO2 / DMI (g / kg) Before experiment 679.32±57.02 685.12±28.64 712.25±32.36 0.363 15 d 714.54 ± 79.21 a ]] 571.34 ± 35.78 b ]] 658.20 ± 92.44 ab ]] 0.013 30 d 751.09 ± 36.52 a ]] 629.06 ± 47.19 b ]] 728.54 ± 80.32 a ]] 0.005 60 d 774.45 ± 36.12 a ]] 668.66 ± 49.24 b ]]> 760.86 ± 39.49 a ]] 0.001

[0084] a,b,c Representative P < 0.05.

[0085] From the application point of view, the 2% addition ratio can not only achieve a significant effect of reducing CH4 emission by 11.4% under the premise of maintaining the normal feeding behavior of animals, but also can simultaneously reduce CO4 emission by 13.7%, showing double environmental benefits. The research results provide important practical basis for developing methane emission reduction technology of ruminants based on Gracilaria addition.

[0086] The present application found through in vitro experiments that using Gracilaria as a fattening beef feed additive can regulate the relative proportion of volatile fatty acids such as acetic acid, propionic acid and butyric acid in the rumen, optimize the fermentation process; at the same time, promote the proliferation of beneficial flora and effectively inhibit the activity of methanogens. In addition, Gracilaria can also induce the synthesis of unsaturated fatty acids such as 13(S)-HOTrE and 9(S)-HOTrE, and then target to block the metabolic pathway of methanogenic archaea, further inhibit its activity. Overall, Gracilaria reduces methane emissions through multiple pathways such as regulating rumen VFA spectrum, remodeling microbial community structure, and inducing synthesis of specific metabolites.

[0087] Finally, it should be explained that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. Application of Gracilaria fusiforme in the preparation of an additive for reducing methane emissions from fattening beef cattle, wherein the additive is added to the feed at an amount of 2 wt%.

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