Application of laurencia or composition of laurencia and seaweed in ruminant animal feeding or ruminant animal feed

Abstract

The invention discloses application of a laurencia or a seaweed composition containing the laurencia as a feed additive in production of reducing methane and carbon dioxide in gastrointestinal tracts of ruminants, and application of the laurencia or the seaweed composition containing the laurencia in preparation of feed with the effect of reducing the methane and the carbon dioxide in the gastrointestinal tracts of the ruminants. The laurel algae combination is a combination of laurel and one or more of the following algae: gulfweed, enteromorpha, kelp, laver and sargassum fusiforme. According to the invention, the laurencia or the laurencia seaweed composition is added into the basal feed, so that the laurencia or the laurencia seaweed composition has a good effect of inhibiting generation of methane and carbon dioxide in gastrointestinal tracts of ruminants. According to the invention, the emission of methane or carbon dioxide of the ruminants is obviously reduced by adding the laurencia or the seaweed combination thereof into the basic ration of the ruminants, the feed utilization rate of the ruminants is improved, and the laurencia or seaweed combination has important value and significance in the aspects of green emission reduction, energy conservation, efficiency improvement and the like of the breeding industry, and has great industrial application potential.

Description

Technical Field

[0001] The invention relates to the application of concave-capped algae and a seaweed combination thereof as a feed additive in reducing gastrointestinal methane emission, belonging to the technical field of ruminant methane emission reduction. Background Art

[0002] Ruminant methane production has an important impact on global carbon dioxide release. Methane is a greenhouse gas with a greenhouse effect 20 times higher than that of carbon dioxide. Although carbon dioxide is the main greenhouse gas, the contribution of methane cannot be ignored. According to the Food and Agriculture Organization of the United Nations (FAO), methane produced by ruminants accounts for about 14.5% of global greenhouse gas emissions. Methane is the second largest greenhouse gas after carbon dioxide, and its anthropogenic emissions contribute about 20% of the greenhouse effect. Agricultural production is the main source of anthropogenic methane emissions. The ruminant breeding process, mainly cattle, contributes 16% of global anthropogenic methane emissions. Methane emissions from animal husbandry mainly come from three aspects: one is the fermentation process of feed stacking; the second is the manure treatment process; and the third is the methane produced by fermentation in the rumen of ruminants and discharged into the atmosphere through rumination. The first two sources can be collected as clean energy by setting up a closed fermentation system, but it is difficult to completely collect methane emitted by rumination during the pastoral process. Therefore, ruminant gastrointestinal methane emission reduction technology is an important topic for methane emission reduction in animal husbandry. At present, there are three technical strategies for reducing methane emissions from the gastrointestinal tract of ruminants. The first is to increase concentrate feed, adjust daily ration nutrition, and improve feed quality; the second is to artificially add probiotics to change the structure of gastrointestinal microbial communities; the third is to specifically add methane inhibitors to feed. The first strategy is costly and the methane emission reduction efficiency is less than 50%. The discontinuous change in the ratio of concentrate to roughage in the feed may also lead to a series of negative hazards such as acute or subacute acidosis of the rumen, disturbance of digestive tract microbial flora, hoof disease, ketosis, etc. The technology of the second strategy is not yet mature, and the long-term effect is unknown. Specially adding methane inhibitors to feed is considered to be the most cost-effective methane emission reduction strategy. Vegetable oils containing unsaturated fatty acids, plant extracts containing tannins and saponins, 3-nitrooxypropanol, nitrates, etc. have been tried as ruminant feed additives, and these substances accumulate in ruminants, causing ruminant growth to be affected, and long-term accumulation in animals causes food safety risks and causes concerns.

[0003] Feeding ruminants with marine algae as feed additives can effectively reduce their methane emissions. Some studies have confirmed that adding an appropriate amount of marine algae can reduce methane emissions from ruminants without significantly affecting their growth and production performance. The active ingredients in marine algae can be extracted and prepared into biofermentation agents to improve the balance of microbial communities in the digestive tract of ruminants and reduce methane production. Euglena gracilis is a plant of the genus Euglena in the family Pinaceae. It is widely distributed in seawater. So far, there has been no report on the use of Euglena gracilis to reduce greenhouse gas emissions from ruminants. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention has found through experimental research that adding concave-capped algae or its seaweed combination to feed can effectively reduce the production of methane and carbon dioxide in ruminants, and play a role in emission reduction, which belongs to the new use category of concave-capped algae. After feeding the combination to ruminants, the methane content in the gastrointestinal tract can be effectively reduced, which plays a good role in inhibiting rumination and reducing the emission of methane and carbon dioxide in the gastrointestinal tract.

[0005] The main purpose of the present invention is to provide a new use of concave-capped algae in ruminant breeding;

[0006] The above object of the present invention is achieved through the following technical solutions:

[0007] The present invention provides a use of concave-capped algae in reducing methane or carbon dioxide emissions of ruminants, comprising: adding the concave-capped algae as a feed additive to the basic diet of the ruminants.

[0008] A preferred specific embodiment of the present invention, for example, is to add the algae powder of Concave Cap Algae to the basal diet of ruminants at an addition amount of 0.1-20wt%; preferably, is to add the algae powder of Concave Cap Algae to the basal diet of ruminants at an addition amount of 0.5-10wt%.

[0009] The ruminants described in the present invention include but are not limited to buffalo, cattle, beef cattle, dairy cows, camels, giraffes, sika deer, sheep, goats, antelopes or alpacas, etc.; preferably buffalo, cattle, beef cattle or dairy cows.

[0010] The concave-capped algae described in the present invention can be any concave-capped algae purchased through commercial channels or concave-capped algae separated by oneself, and all of them are applicable to the present invention.

[0011] The present invention is fully mixed Daily DietAs the basic diet, the algae powder of the concave top algae was added to the basic diet of dairy cows at an addition amount of 1% and 2% of the dry weight of the substrate, respectively. Through the in vitro fermentation test of the rumen of beef cattle, it was found that the algae powder of the concave top algae can reduce the proportion of methanogens in the rumen, reduce the methane emissions of the rumen of beef cattle by 16.37%, and reduce the carbon dioxide emissions of the rumen of beef cattle by 27.62%; in addition, it was also found that the addition of the algae powder of the concave top algae can also reduce the concentration of volatile fatty acids. The present invention determines that adding concave top algae to the basic diet of ruminants can reduce the methane or carbon dioxide emissions of ruminants and improve the feed utilization rate of ruminants, which has important application prospects for green emission reduction, energy saving and efficiency improvement in the breeding industry.

[0012] The present invention adds concave-cap algae or a concave-cap algae seaweed combination to the basic feed, which has a good effect of inhibiting the production of methane and carbon dioxide in the gastrointestinal tract of ruminants. The raw materials used in the present invention are widely available, low in cost, and have a significant effect of inhibiting the production of methane and carbon dioxide in the gastrointestinal tract. The present invention significantly reduces the methane or carbon dioxide emissions of ruminants and improves the feed utilization rate of ruminants by adding concave-cap algae or a combination of seaweeds to the basic diet of ruminants. It has important value and significance in green emission reduction, energy saving and efficiency improvement in the aquaculture industry, and has great potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 .Effects of supplementation with Concavoglossum on in vitro gas production and gas composition in beef cattle. LAT: supplemented with 0.5% Concavoglossum; HAT: supplemented with 2% Concavoglossum; * indicates a significant difference between HAT and CON (P<0.05); ★ indicates a significant difference between LAT and CON (P<0.05); Figure A: total rumen gas production in 48 hours; Figure B: 48-hour dietary dry matter degradation rate; Figure C: ratio of carbon dioxide production in 48 hours to total gas production; Figure D: ratio of hydrogen production in 48 hours to total gas production; Figure E: ratio of methane production in 48 hours to total gas production. DETAILED DESCRIPTION

[0014] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0015] Example 1: Study on reducing methane or carbon dioxide emissions from beef cattle by using concave-capped algae

[0016] In this example, the methane and carbon dioxide emission reduction effects of the concave-capped algae were detected by in vitro culture of the rumen gastric fluid of adult beef cattle. The specific process is as follows:

[0017] 1) All animal experiments were approved by the Animal Welfare and Ethics Committee of the Institute of Oceanology, Chinese Academy of Sciences (No: IOAS2022-92). Concave algae: collected from the South China Sea and identified as Concave algae by the Classification Office of the Institute of Oceanology, Chinese Academy of Sciences. About 1L of rumen fluid was collected from the rumen fistula of three Simmental cross bulls before feeding in the morning, and the average weight of each bull was 500 kg. The rumen fluid was filtered through 4 layers of gauze (60 mesh) and evenly mixed for use. A total of three diets were designed in this study, including one basic diet and two Concave algae supplemented diets. The basic diet (Table 1) served as the control group (CON). In the experimental diet (Table 1 basic diet), 0.5% or 2% of the corn silage mass of the basic diet was added to CON and replaced by Concave algae (by dry weight), and the two Concave algae supplemented diets were obtained as 0.5% AT and 2% AT groups, respectively. The composition of each diet is shown in Table 1.

[0018] Table 1. Feed ingredients and chemical composition of the basal diet (per dry matter)

[0019]

[0020] 2) Accurately weigh 200 mg of each diet (basal diet, 0.5% AT and 2% AT supplemented diet) and place it into a 100 mL glass syringe equipped with a plunger. The syringe was filled with a mixed solution containing 10 mL rumen fluid and 20 mL PBS buffer solution. There were 6 replicates for each diet, and the other 3 replicates were used as blanks without dietary additions. All glass syringes were placed in a biochemical incubator (SHP205; Peiyin, Shanghai, China) at 39°C. The gas production (GP) was monitored and recorded at 2, 4, 6, 8, 12, 24, and 48 h of incubation. After 48 h of incubation, culture fluid samples were collected for volatile fatty acid (VFA) analysis, and gas samples were collected to evaluate the concentrations of CO2, H2, and CH4. In addition to the gas production test, 500 mg of the basal diet group, 0.5% and 2.0% A. conchae supplemented diet were added to a 50 mL centrifuge tube, 10 mL of rumen fluid and 20 mL of PBS buffer solution were added for incubation. The residuals after 48 h of fermentation were collected to measure the dry matter degradability, and each treatment was repeated 5 times.

[0021] The results are shown in Table 2 and Figure 1As shown, adding 0.5% or 2.0% of Concave Top Algae to the basal diet significantly reduced the rumen gas production of beef cattle. The soluble fraction gas production of the Concave Top Algae components (0.50% AT and 2.00% AT groups) added 0.5% or 2.0% decreased by 34.78% and 73.91% respectively compared with the control group (CON) of the normal diet; the insoluble fraction gas production was compared with the control group (CON), and the 0.50% AT and 2.00% AT groups decreased by 14.11% and 19.17% respectively. The gas production rate constant (% / h) decreased by 19.32% and 32.95% respectively in the 0.50% AT and 2.00% AT groups compared with the control group (CON). The results show that adding 0.5% or 2.0% of Concave Top Algae to the basal diet can significantly inhibit the rumen gas production of beef cattle. Further research analyzed the effect of supplementing concave algae on the total gas production of beef cattle. The results are shown in Table 3. Compared with the control group, the total gas production of beef cattle rumen in 48 hours decreased from 339.32mL / h to 296.28mL / h and 265.41mL / h, respectively, a decrease of 12.68% and 21.80%. Among them, the methane (CH4) gas production decreased by 20.93% and 52.25% compared with the Con group 0.50% AT and 2.00% AT groups; the carbon dioxide (CO2) gas production decreased by 27.83% and 38.90%; the hydrogen (H2) gas production decreased by 46.423% and 53.57%. And the decrease in gas production did not significantly affect the degradation rate of dry matter (DM). The results showed that adding 0.50% or 2.00% of Concave-top Algae to the diet could significantly inhibit the gas production of beef cattle, and had a significant inhibitory effect on the gas production of methane, carbon dioxide and H2. In particular, it had a very significant inhibitory effect on methane and carbon dioxide, the main gases produced by beef cattle digestion, and the inhibitory effect of 2.00% Concave-top Algae on gas production was better than that of 0.50% Concave-top Algae.

[0022] Table 2 Effects of supplementation with Algae on dynamic parameters of in vitro gas production in beef cattle

[0023]

[0024] Table 3. Effect of supplementation with concave algae on total gas production of beef cattle

[0025]

[0026] Figure 1 Effects of supplementation with Concavoglossum on in vitro gas production and gas composition in beef cattle. LAT: supplemented with 0.5% Concavoglossum; HAT: supplemented with 2% Concavoglossum; * indicates a significant difference between HAT and CON (P<0.05); ★ indicates a significant difference between LAT and CON (P<0.05).

[0027] 3) Volatile fatty acids and gas composition analysis

[0028] The rapid accumulation of volatile fatty acids (VFA) can easily cause a decrease in rumen pH and lead to rumen acidosis. Therefore, the effect of adding concave top algae on rumen volatile fatty acids was investigated in this example. The specific experimental operation was as follows: After the rumen fluid was incubated with different diet components for 48 hours, the glass syringe was placed on ice to terminate the microbial fermentation. About 5 mL of total gas in each glass syringe was collected, and the concentrations of CO2, H2, and CH4 were immediately determined using gas chromatography (GC-8600; FTIR, Beijing, China). In addition, 10 mL of culture medium was collected and stored at -20 ° C for future DNA extraction and volatile fatty acid (VFA) analysis. Gas chromatography (GC-8600; FTIR, Beijing, China) was used. To prepare samples for VFA analysis, 1 mL of culture medium was mixed with 250 μL of 25% H3PO3 by mass concentration, and the mixture was centrifuged at 15,000 × g and 4 ° C for 15 minutes. Subsequently, 5 μL of supernatant was injected into the gas chromatograph to evaluate the VFA spectrum. The injector temperature was set to 200° C. The column temperature was increased from 45° C. to 130° C. at a rate of 10° C. / min and then maintained at 130° C. for 20 min.

[0029] The results are shown in Table 4. Supplementing the diet with concave-capped algae can increase the content of effective fiber in the feed and reduce the concentration of volatile fatty acids after rumen digestion (mmol / L). Among them, the total volatile fatty acids added with 2.00% concave-capped algae in the diet decreased from 110.26mmol / L in the control group (Con) to 85.08mmol / L, significantly reducing the production of total volatile fatty acids.

[0030] Appendix 4: Effects of supplementation with Acanthocephalum on volatile fatty acid concentrations (mmol / L)

[0031]

[0032] Study on the effect of Algae sphenanthera on optimizing rumen microbial flora

[0033] 4) Analysis of rumen gas-producing bacteria

[0034] The composition of rumen microorganisms affects rumen rumination and the production of methane and carbon dioxide. Therefore, in the experiment of this example, targeted amplicon sequencing and bioinformatics were used to analyze the composition of rumen microorganisms before and after feeding Concavocapra.

[0035] The specific experimental operation is as follows: After the diet supplemented with A. concavitas was incubated with rumen fluid for 48 hours, the culture fluid (repeated times n = 6) was taken to extract DNA, and further targeted amplicon sequencing was performed to analyze the changes in rumen microbial composition based on the effects of different microorganisms on CH4 production. DNA was extracted using the Magnetic Soil and Stool DNA Kit (TianGen, China). 357F (5'-ACTCCTACGGRAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were used to amplify the V3-V4 region of 16S rRNA. All PCR reactions used 15 μL High-Fidelity PCR master mix was used. PCR products were mixed at equal concentrations and purified using the Universal DNA Purification Kit (TianGen, China). NEB Ultra TMII FSDNA free Library Prep Kit (New England Biolabs, USA) was used to generate sequencing libraries and add indexes. Libraries were checked for quantification using Qubit and real-time PCR according to the manufacturer's instructions, and indexes were added. Libraries were checked for quantification using Qubit and real-time PCR, and size distribution was detected using a bioanalyzer. Quantitative libraries were pooled and sequenced on an Illumina Miseq sequencing platform with a 250 bp double-end read length. Sequenced double-end reads were assigned to samples according to their unique barcodes, and their barcodes and primer sequences were removed for truncation using Python (V3.6.13). Adapters were removed using Cutadapt (V3.3), and high-quality clean reads were obtained using 0.23.1) (Bokulich, Subramanian et al. 2013). Clean reads were aligned to the Silva database (https: / / www.arb-silva.de / ) using the UCHIME algorithm (http: / / www.drive5.com / usearch / manual / uchime_algo.html) to detect chimeric sequences. Valid reads were obtained by removing chimera sequences using the vsearch (V2.16.0) package (Edgar, Haas et al. 2011). Afterwards, sequence analysis was performed using Uparse software (Uparsev7.0.100, http: / / drive5.com / uparse / ) (Edgar and Robert 2013), and sequences with a similarity of ≥97% were assigned to the same operational taxonomic unit (OTU). OTU representative sequences were classified and annotated into phylum, class, order, family, genus, and species using the BLAST online software in the Silva 128 database.

[0036] The results are shown in Table 5, indicating that the addition of 0.5% or 2.0% of Concavocaps (0.5% AT or 2.0% AT) to the basal diet can significantly reduce the content of Methanobacteria in the rumen; at the same time, it also has a significant inhibitory effect on microorganisms of the genera Clostridium and Morgibacter. It has an up-regulating effect on the NK4A136 group of Lachnospiraceae, the anaerobic Vibrio genus, and the ND3007 group of Lachnospiraceae. Therefore, the results show that the addition of 0.5% or 2.0% of Concavocaps to the basal diet can improve the distribution of rumen microbial gas-producing bacteria and optimize the rumen microbial environment.

[0037] Table 5. Effects of dietary supplementation with Algae on the rumen microbiota of beef cattle compared with the control diet

[0038]

Claims

1. Use of concave-capped algae or a combination of concave-capped algae and seaweed in ruminant breeding or ruminant feed.

2. The use according to claim 1, characterized in that: The types of the concave-capped algae include one or more of Laurencia okamurai, Laurencia tristicha, and Chondrophycus undulatus.

3. The use according to claim 1, 2 or 3, characterized in that : The seaweed includes one or more of Sargassum, Enteromorpha, Laminaria, Porphyra, and Sargassum fusiformis; in the combination of concave-capped algae and seaweed, the mass proportion of concave-capped algae in the seaweed combination is greater than or equal to 20%, preferably 20-50%, and more preferably 30-40%.

4. The use according to any one of claims 1 to 3, characterized in that: include: The concave-capped algae or the concave-capped algae and seaweed combination is added into the basic diet of ruminants as a feed additive.

5. The use according to claim 1, 2 or 3, characterized in that: The product form of the concave-capped algae and the seaweed combination thereof is any one of fresh product, dry product or freeze-dried powder.

6. The use according to claim 1, 4 or 5, wherein the amount of the concave-capped algae or the combination of concave-capped algae and seaweed added to the feed is 0.5% to 10%, calculated on a dry weight basis, in weight percentage.

7. The use according to claim 1, 2 or 3, characterized in that: The preparation process is: Take concave-capped algae, or concave-capped algae and one or more of sargassum, enteromorpha, kelp, laver, and fusiforme, mix and grind them, and pass them through a 40-60 mesh sieve to obtain a product that passes through the sieve.

8. The use according to any one of claims 1 to 7, characterized in that: It can reduce methane and / or carbon dioxide emissions from ruminants; The ruminants include but are not limited to buffalo, cattle, beef cattle, dairy cows, camels, giraffes, sika deer, sheep, goats, antelopes or alpacas.

9. A ruminant feed having the effect of reducing methane production in the gastrointestinal tract of ruminants, characterized in that: The invention is composed of basic feed and concave-capped algae or a combination of concave-capped algae and seaweed, wherein the addition amount of the concave-capped algae or the combination of concave-capped algae and seaweed in the feed is 0.5% to 10%, calculated on dry weight, weight percentage.

10. The feed according to claim 9, characterized in that: The mass proportion of concave-capped algae, or the combination of concave-capped algae and seaweed, in the seaweed combination is not less than (greater than or equal to) 20%, preferably 20-50%, more preferably 30-40%.

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