Feed for improving immune function of cattle and application thereof

Abstract

The invention relates to the technical field of livestock and poultry breeding, in particular to feed for improving the immune function of cattle and application of the feed. According to the method, the influence of feeding different proportions of oat hay and alfalfa hay on the growth performance and the immune function of the calf is researched, and the relationship of rumen microorganisms and cecum microorganisms on the production performance and the immune function is clarified by analyzing rumen microbiome and posterior intestinal microbiome. The antioxidant performance of the calves can be influenced by improving intestinal microflora by increasing the proportion of the alfalfa in the daily ration, and meanwhile, the growth performance of livestock can be improved by mixing the oat hay.

Description

Technical Field

[0001] This invention relates to the field of livestock and poultry breeding technology, and in particular to a feed for improving the immune function of cattle and its application. Background Technology

[0002] Between 2020 and 2050, global average protein demand for red meat, poultry, milk, and eggs will increase by 14%, with total demand increasing by 38%. Lamb is a significant component of global red meat consumption. The most important factors influencing calf productivity include host genetics (breed), sex, growth stage or age, dietary factors, environmental factors, and feeding methods. Compared to monogastric animals, ruminants such as cattle, sheep, and beef cattle utilize fiber more effectively. Adding appropriate amounts of fiber to the diet can stimulate chewing and rumination in sheep, promote saliva secretion, enhance rumen fermentation, and improve the quality of livestock products. However, excessive crude fiber content in the diet can affect palatability and reduce dry matter intake in sheep. Therefore, finding a strategy for selecting high-quality roughage is crucial for improving calf productivity and meeting red meat requirements.

[0003] Feeding high-quality roughage is beneficial for maintaining rumen homeostasis and reducing the occurrence of metabolic diseases such as rumen acidosis and abomasal displacement. Currently, high-quality forages such as alfalfa, oat grass, and ryegrass are major sources of high-quality protein in livestock production and provide animals with various vitamins and minerals. Alfalfa, as a high-quality forage with strong stress resistance, has multiple characteristics such as promoting digestion and absorption in ruminants and maintaining their health, making it one of the most popular roughages in the world. Meanwhile, because oat grass has lower levels of neutral detergent fiber and lignin than alfalfa and other roughages, it has good palatability, helping to improve the digestion and absorption of ruminants and feed utilization efficiency, and is gradually becoming an important roughage source in my country's livestock production. The combination of oats and alfalfa may provide fattening sheep with energy and effective physical fiber, ensuring growth efficiency while protecting rumen health.

[0004] The synergistic supply of alfalfa hay, starter cultures, and milk replacers significantly improved rumen fermentation capacity in cattle and lambs, altering the rumen microbiota, which was a major reason for the improved growth performance in both animals (Yang et al., 2018; Wu et al., 2019a). In addition to rumen fermentation, the addition of alfalfa hay can also affect rumen pararumen starch and fiber, thereby influencing the hindgut microbiota and fermentation (Qiu et al., 2019). As the main hindgut, cecal fermentation is also a crucial factor affecting growth performance; changes in cecal microbiota lead to improved immune function. However, the mechanism by which changes in roughage affect immune function through alterations in the gut microbiota remains unclear. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a feed for improving bovine immune function and its application. This invention explores the effects of different mixing ratios of alfalfa and oats on the production performance and immune function of calves, and elucidates the relationship between rumen microorganisms and cecal microorganisms on production performance and immune function.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a feed to enhance the immune function of cattle, comprising alfalfa hay and oat hay.

[0008] Preferably, the mass ratio of alfalfa hay to oat hay is 3-7:3-7.

[0009] Preferably, the alfalfa hay includes alfalfa.

[0010] The present invention also provides the application of the feed described in the above technical solution in the preparation of products that improve bovine immune function.

[0011] The present invention also provides the application of the feed described in the above technical solution in the preparation of products that improve bovine production function.

[0012] The present invention also provides the feed or application described in the above technical solution, wherein the cattle include calves.

[0013] The beneficial effects of this invention are:

[0014] This invention investigates the effects of feeding different ratios of oat hay and alfalfa hay on the growth performance and immune function of calves. By analyzing the rumen and hindgut microbiomes, the relationship between rumen and cecal microbiomes on production performance and immune function is elucidated. In a 70-day experiment, 32 calves were fed oat hay, oat-alfalfa (3:7 ratio), oat-alfalfa (7:3 ratio), and alfalfa (8 calves per group). Results showed that feeding all alfalfa significantly reduced the daily weight gain of fattening calves, decreasing by 19.21% compared to the highest ratio. Although feed intake did not change significantly, the feed conversion ratio in the all-alfalfa group was significantly lower, ranging from 30.24% to 36.47%. However, with increasing alfalfa content, TAOC and MDA showed a trend, and the antioxidant capacity of fattening calves fed with high alfalfa content was significantly higher than that of the low alfalfa content group. Importantly, the abundance of hindgut microbes Bacteroidales_unclassified and Clostridium were strongly correlated with TAOC and MDA. Therefore, increasing the proportion of alfalfa in the diet can affect the antioxidant performance of calves by improving the gut microbiota, while mixing oat hay helps improve livestock growth performance. Attached Figure Description

[0015] Figure 1 This is the PCoA map. Detailed Implementation

[0016] This invention provides a feed to enhance bovine immune function, comprising alfalfa hay and oat hay. In this invention, the preferred mass ratio of alfalfa hay to oat hay is 3-7:3-7. In this invention, the alfalfa hay preferably includes alfalfa. This invention does not specifically limit the preparation method of the feed; simply mixing the alfalfa hay and oat hay is sufficient. In this invention, the bovines preferably include calves.

[0017] This invention also provides the application of the feed described in the above-described technical solution in the preparation of products that enhance bovine immune function. In this invention, the bovine preferably includes calves.

[0018] This invention also provides the application of the feed described in the above-described technical solution in the preparation of products that improve bovine productivity. In this invention, the bovine preferably includes calves.

[0019] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1

[0021] 1. Materials and Methods

[0022] 1.1 Experimental Design

[0023] This experiment was conducted at the Acheng Experimental Base of Northeast Agricultural University from February to April 2024. Thirty-two healthy, disease-free male Holstein calves at 6 months of age with an average weight of 130.63 ± 0.20 kg were selected as experimental subjects and randomly divided into four groups based on similar weights, with eight replicates per group. Whole oats, oats, and alfalfa were all used as hay. The experimental groups were treated with whole oats (OAT), oats:alfalfa (OA) = 7:3, oats:alfalfa (OA) = 3:7, and whole alfalfa (ALFALFA), respectively. All calves were individually housed with free access to water. A concentrate-roughage separation feeding method was adopted, with a concentrate-roughage ratio of 65:35, and calves were fed at 06:00 and 18:00 daily. The experiment lasted for 70 days, including a 14-day pre-feeding period and a 56-day experimental period.

[0024] 1.2 Composition and nutrient levels of the experimental diet

[0025] The diet consists of concentrates such as corn, corn germ meal, sprayed corn husks, DDGS and extruded soybeans, as well as roughage mixed with alfalfa and oat grass in different proportions. The composition and nutritional level of the diet are detailed in Table 1.

[0026] Table 1. Dietary composition and nutrient levels (%, percentage by mass)

[0027]

[0028]

[0029] 1.3 Sample collection and data recording

[0030] After a 14-day acclimatization period, a 56-day feeding trial began. Every 7 days, feed ingredients and leftovers from each group of experimental calves were collected and mixed evenly. The collected feed samples were dried in a 55°C oven to constant weight. The dried samples were pulverized through a 1mm sieve, labeled, and sealed for storage at -20°C. The collected feed samples and leftovers were used to determine their chemical composition.

[0031] On days 51, 52, and 53 of the experimental period, a digestibility test was conducted using the full fecal collection method. Approximately 500g of fresh hind intestine samples were collected each morning at 7:00 AM. 5% of the hind intestine was taken from each calf. The samples from the three consecutive days were mixed thoroughly and divided into two equal portions. For one portion, 100g of hind intestine was mixed with 10mL of 10% hydrochloric acid for nitrogen fixation. Both portions of hind intestine samples were dried in a 55℃ oven to constant weight. The dried samples were then pulverized using a 1mm sieve, labeled, and sealed for storage at -20℃ for determining nutrient digestibility.

[0032] On day 56 of the experiment, 2 hours after morning feeding, 50 mL of rumen fluid was collected using the oral tube collection method. In order to reduce the dilution of rumen fluid by saliva, the first sample was discarded before each sampling. The filtrate was collected after being filtered through four layers of gauze.

[0033] 1.4 Index Measurement

[0034] 1.4.1 Determination of growth performance

[0035] On days 0 and 56 of the experimental period, calves in each group were weighed before morning feeding, and their weight was recorded to calculate the average daily gain (ADG). From the start of the formal experiment, the amount of feed and leftover feed for each group of calves was recorded daily in detail, and total feed intake and feed efficiency were calculated using the following formula:

[0036] Average daily weight gain (g) = Final weight of experimental calves - Initial weight of experimental calves / Number of experimental days

[0037] Total feed intake (kg) = Total feed intake during the trial period - Total feed residue during the trial period

[0038] Feed efficiency (%) = Total feed intake of experimental calves / (Final weight of experimental calves - Initial weight of experimental calves)

[0039] 1.4.2 Determination of feed composition and apparent digestibility

[0040] According to the procedures of the Association of Analytical Chemists, the dry matter (DM), crude protein (CP), and crude fat (EE) contents in feed ingredient samples, leftover feed samples, and hindgut samples were determined by wet chemical analysis in the Ruminant Nutrition Laboratory of Northeast Agricultural University. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were analyzed according to the method of Van Soest et al. [Soest PJ V. Nutritious Ecology of the Ruminant[J]. Cornell University P,1994,44(11):2552-2561.DOI:10.2514 / 1.18760.]. The results of NDF and ADF are expressed on a dry matter (DM) basis. Apparent nutrient digestibility was determined based on hydrochloric acid insoluble ash (AIA) in feed and hindgut samples, and calculated according to the method of the standard "Determination of Hydrochloric Acid Insoluble Ash in Feed" (GB / T 23742-2009).

[0041] 1.4.3 Rumen Microbiome and Hinter Gut Microbiome

[0042] Rumen and hindgut samples were analyzed for 16S rDNA sequencing at LC-Bio Technology Co., Ltd. A hindgut DNA kit (D4015, Omega, Inc., USA) was used. Nucleus-free water was used as a blank. Total DNA was eluted with 50 μl of elution buffer and stored at -80°C until polymerase chain reaction (PCR) analysis at LC-Bio Technology Co., Ltd. (Zhejiang, China). The 5' ends of the primers were labeled with dedicated barcodes and universal sequencing primers. PCR amplification was performed using 25 ng of template DNA, 12.5 μl of PCR Premix, 2.5 μl of each primer, and 25 μl of PCR-grade water, adjusting the final volume accordingly. PCR conditions for amplifying the prokaryotic 16S fragment were as follows: initial denaturation at 98°C for 30 s; 32 cycles of denaturation at 98°C for 10 s, annealing at 54°C for 30 s, extension at 72°C for 45 s; and final extension at 72°C for 10 s. PCR products were confirmed by 2% agarose gel electrophoresis. Ultrapure water was used as a negative control throughout the DNA extraction process to eliminate the possibility of false positives in PCR results. PCR products were purified using AMPureXT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Amplicon libraries were prepared for sequencing, and the size and number of amplicon libraries were assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, USA) and the KAPA library quantification kit (KAPA Biosciences, Woburn, MA, USA) on the Illumina sequencing platform, respectively. These libraries were sequenced on a NovaSeq PE250 platform.

[0043] 1.4.5 Statistical Analysis

[0044] Growth parameter data were analyzed using one-way ANOVA with Duncan's multiple range test. Alpha diversity index data are expressed as mean ± standard deviation and analyzed using Fisher's least significant difference (LSD) test with ANOVA. Statistical analysis was performed using SPSS version 25.0 on Windows (SPSS Inc., Chicago, Illinois, USA). A p-value < 0.05 was considered statistically significant.

[0045] Following the manufacturer's recommendations (LC-Bio Technologies), samples were sequenced on the Illumina NovaSeq platform. Reads were paired end-to-end based on the sample's unique barcode, and truncated by cutting the barcode and primer sequences. End-to-end reads were merged using FLASH. Raw reads were quality filtered using fqtrim (v0.94) under specific filtering conditions to obtain high-quality clean labels. Chimeric sequences were screened using VSEARCH software (v2.3.4). After deduplication using DATA2, a feature table and feature sequences were obtained. Beta diversity was calculated by randomly removing aligned reads and normalizing to the same sequencing depth. Feature abundance was then normalized using the relative abundance of each sample according to the SILVA (release 132) classifier. Beta diversity was calculated using QIIME2 and plotted using R packages. A Spearman correlation matrix was plotted using R packages (v3.6.3) to analyze the correlation heatmap. Sequence alignment was performed using Blast, and each representative sequence was labeled with the SILVA database. The other charts were generated using the R package (v3.5.2).

[0046] result

[0047] The results showed the growth performance of different proportions of roughage. An analysis of variance was conducted with initial body weight as a covariate. The results showed that feeding whole alfalfa significantly reduced the daily weight gain of fattening calves. Compared with the highest group, the daily weight gain decreased by 19.21%. Although feed intake did not change significantly, the feed conversion ratio of the whole alfalfa group was significantly reduced by 30.24% to 36.47%.

[0048] Table 2 Results Data

[0049] project Oat OA7:3 OA3:7 Alfalfa SME P-value Initial weight, kg 131.80 132.26 132.88 129.40 1.33 0.909 Final weight, kg <![CDATA[146.16 a ]]> <![CDATA[149.68 a ]]> <![CDATA[152.30 a ]]> <![CDATA[142.98 b ]]> 1.29 <0.001 Feed conversion ratio, kg / kg <![CDATA[62.52 a ]]> <![CDATA[65.38 a ]]> <![CDATA[65.45 a ]]> <![CDATA[85.26 b ]]> 3.90 0.033

[0050] In this embodiment, the results of nutrient metabolism show that, overall, with the increase of alfalfa content, the digestibility of all nutrients except fat digestibility is significantly improved. Compared with whole oats, whole alfalfa has an 8.25% increase in CP digestibility, a 23.39% increase in NDF digestibility, a 37.14% increase in ADF digestibility, and a 14.92% increase in dry matter digestibility.

[0051] In the analysis of immune function, live weight was used as a covariate in this embodiment. The results showed that the slaughter rate of fattening calves fed OA7:3 was significantly lower than that of other groups. As the alfalfa content increased, TAOC and MDA showed trend changes. Calves fed with high alfalfa content had significantly higher antioxidant capacity than those fed with low alfalfa content.

[0052] Table 3 Data Results

[0053] project Oat OA73 OA37 Alfalfa SME P-value T-AOC (nmol / ml) 1.33 1.89 4.08 3.25 0.14 0.050 MDA (nmol / ml) 4.20 3.74 3.64 3.37 0.07 0.035

[0054] To investigate the impact of alfalfa hay and oat hay diet mixtures on beta diversity, this study used unweighted UniFrac distance to characterize the bacterial communities of all rumen samples. PCoA mapping showed that the bacterial community composition of these groups was not significantly segregated. Taxonomic analysis identified 25 phyla and 362 genera in the rumen and 22 phyla and 369 genera in the hindgut. At the rumen phylum level, seven phyla were designated as detectable phyla (relative abundance >1% in at least one group). The most abundant phylum was Firmicutes, followed by Bacteroidetes and Proteobacteria, with a total abundance exceeding 90%, but no significant differences were observed between groups (P>0.05). At the genus level, 26 genera were considered detectable (relative abundance >1% in at least one group). The main genera are Prevotella_1, Muribauculaceae_unclassified, Ruminococcus_2, Succiniclasticum, Veillonellaceae_UCG-001, Rikenellaceae_RC9_gut_group, Succinivibrio, Ruminococcus_NK4A214_group, Christensenellaceae_R-7_group, Prevotella, Prevotellaceae_UCG-001, F082_unclassified, and Lachnospiraceae_unclassified, with a total abundance of 13 genera exceeding 60%.

[0055] At the hindgut level, Firmicutes and Bacteroidetes were the most abundant, with a total abundance exceeding 87%. Spirochaetes, Actinobacteria, Proteobacteria, Verrucomicrobia, and Epsilonbacteraeota all had abundances exceeding 1%. There were no significant differences in the phylum level among the different groups. The main genera were Ruminococcaceae_UCG-005, Rikenellaceae_RC9_gut_group, Christensenellaceae_R-7_group, Eubacterium_coprostanoligenes_group, Lachnospiraceae_unclassified, Ruminococcaceae_UCG-010, Firmicutes_unclassified, Alistipes, Bacteroides, Ruminococcus_1, Treponema_2, Ruminococcaceae_UCG-014, Ruminococcaceae_UCG-013, and Clostridium, with a total abundance exceeding 60%.

[0056] In the genus-level significance results, this example compared the genus-level differences in the microbiota of the alfalfa group and the oat group. The results showed that the Rikenellaceae_RC9_gut_group in the rumen and hindgut was significantly higher than that in the oat group. The Bacteroidales_unclassified in the hindgut of the alfalfa group was significantly higher than that in the oat group. There were no significant changes in the genus level of other rumen microbiota.

[0057] discuss

[0058] This invention demonstrates the effects of different oat and alfalfa mixing ratios on the growth and slaughter performance of finishing calves. The results show that a high proportion of alfalfa in the roughage diet of calves during the finishing stage improves the antioxidant capacity of beef, but consuming only alfalfa reduces daily weight gain and feed conversion ratio. Similar to other studies, complete alfalfa intake in the diet does not significantly improve daily weight gain in livestock, but the Pordomingo study also found that alfalfa can improve the immune function of livestock. Importantly, in this study, oat supplementation with alfalfa improved the daily weight gain of calves.

[0059] Live weight is an important economic trait in livestock farming, and in China, it is the most significant factor influencing market prices. Therefore, daily weight gain and feed conversion ratio directly determine farmers' income. In this study, the daily weight gain and feed conversion ratio of the all-alfalfa group were significantly lower than those of other groups, indicating that adding alfalfa hay alone to the diet cannot effectively convert into weight gain, while the diet mixed with oats can further improve the animals' digestive function. Castells (2016) found that calves fed oats had an average daily weight gain 63% higher than the alfalfa diet group. Interestingly, the results of this invention are consistent with those of Doran (2007), where alfalfa digestibility was significantly higher than that of the oat diet group. Oats are considered to contain more non-structural carbohydrates and have a higher pectin content than alfalfa, which can be rapidly degraded and absorbed into energy in the rumen. However, growth performance is also affected by factors such as climate and feeding methods, and more dry matter may be converted into feces, urine, and other energy loss forms.

[0060] The core phylum-level microbiota consists of Bacteroidetes, Firmicutes, and Proteobacteria, accounting for approximately 90% of bacterial species (Anderson et al., 2017; Guo et al., 2020; Chai et al., 2021). Bacteroidetes and Firmicutes are the most abundant and active phyla involved in carbohydrate and protein degradation (Xue et al., 2020). The main function of Bacteroidetes is to degrade various plant polysaccharides, improve the host's nutrient utilization, and enhance the host's immunity (Bayliss and Houston, 1984). (Scharen et al., 2017; Li et al., 2020a). Firmicutes play another important role in fiber and cellulose degradation, and are involved in the breakdown of polysaccharides and energy utilization (Crisol-Martínez et al., 2017; Liang et al., 2021). In this study, there were no significant differences among the groups at the rumen phylum level, and changes in roughage did not alter bacterial richness and taxonomic composition, but there were different relative abundances at the genus level. This finding is consistent with other reports (Scharen et al., 2017; Li et al., 2020a), which may reflect a specific ecological niche associated with dietary fiber digestion (Shen et al., 2020).

[0061] Many studies have reported that increasing the proportion of alfalfa in the diet can improve the meat quality of animals. This invention confirms that increasing alfalfa content significantly increases the total taurine organic carbon (TAOC) and reduces the malondialdehyde (MDA) content in veal. This result is consistent with the findings of SU (2022). Adding alfalfa meal to Tibetan diets significantly reduced malondialdehyde (MDA) content by 16.5%. The antioxidant free radical scavenging groups in the structures of plant active ingredients such as flavonoids, saponins, and polysaccharides can react with free radicals by providing hydrogen, thus scavenging free radicals. This reduces the raw materials for free radical synthesis and increases enzymes that decompose free radicals, thereby exerting an antioxidant effect. Flavonoids, saponins, and polysaccharides have all shown the effect of increasing TAOC and reducing MDA content in different experimental animals such as dairy cows and mice. This suggests that the combined effect of plant active ingredients in alfalfa may be an important reason for improving the body's antioxidant defense system.

[0062] This invention explored the correlation between hindgut and rumen microbiota and growth and slaughter performance, revealing that in vivo microbiota can influence the antioxidant capacity of calf muscle. *Bacteroidales_unclassified* and *Bacteroidales* were considered to be significantly or extremely significantly positively correlated with TAOC in this study. *Bacteroidales_unclassified* was significantly higher in the alfalfa group than in the oat group, suggesting that *Bacteroidales_unclassified* may be key to improving the antioxidant capacity of meat in alfalfa diets. *Bacteroidales* is well known to play a crucial role in rumen carbon conversion. Although *Bacteroidales_unclassified* lacks a fully defined genome, its biological function may be similar to most other members of the Bacteroidetes family, possessing the ability to release hemicellulose monomeric sugars. Solden's genomic metabolic analysis revealed multiple pathways for fermenting hemicellulose monomeric sugars into short-chain fatty acids (SCFAs), particularly increased butyrate content, which is an important pathway for enhancing antioxidant capacity in the hindgut environment.

[0063] Clostridium and MDA were significantly correlated in the hindgut. In this study, the hindgut clostridium levels in the alfalfa group were significantly lower than those in the oat group. Similarly, Wu's addition of alfalfa hay to the yak diet significantly reduced Clostridium XVIII and LPS levels, decreased inflammatory response, and improved liver health in yaks. Increased clostridium levels in the gut can induce clostridial abomasitis and enteritis, characterized by necrosis of eubacterial bacteria or intestinal mucosa caused by exotoxins produced by Clostridium perfringens or Clostridium difficile in the gastrointestinal lumen. The proliferation of Clostridium perfringens in the gastrointestinal tract of ruminants is associated with increased carbohydrate or protein intake and altered gastrointestinal motility (Simpson 2017). In this study, a high-concentrate diet may have increased clostridium abundance, while the addition of alfalfa hay reduced clostridium abundance, which is the reason for the decrease in MDA. Furthermore, the results of this invention show that the pH of veal meat is significantly correlated with Olsenella in the rumen and Rikenellaceae_RC9_gut_group in the gut, which means that different proportions of alfalfa and oats alter the body's microbiota and thus affect immune function.

[0064] in conclusion

[0065] A high proportion of alfalfa in roughage for calf growth diets increases blood antioxidant capacity, but whole alfalfa reduces daily weight gain and feed conversion ratio. Alfalfa feeding increases the abundance of *Bacteroidales* unclassified and decreases the abundance of *Clostridium*, and these microbiomes are strongly correlated with calf antioxidant capacity. Different roughage compositions affect the gut microbiota, thereby improving the immune function of young ruminants.

[0066] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A feed for improving bovine immune function, characterized in that, This includes alfalfa hay and oat hay.

2. The feed according to claim 1, characterized in that, The mass ratio of alfalfa hay to oat hay is 3-7:3-7.

3. The feed according to claim 1, characterized in that, The alfalfa hay includes alfalfa.

4. The use of the feed according to any one of claims 1 to 3 in the preparation of products that enhance bovine immune function.

5. The use of the feed according to any one of claims 1 to 3 in the preparation of products that improve bovine productivity.

6. The feed according to any one of claims 1 to 3 or the application according to claim 4 or 5, characterized in that, The cattle include calves.

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