Application of guar gum and xylo-oligosaccharide in promoting the growth of sika deer antler

Abstract

The application discloses application of guar gum and xylo-oligosaccharide in promoting growth of sika deer antler and belongs to the technical field of animal breeding.The application first discloses that the guar gum and the xylo-oligosaccharide promote the growth of the sika deer antler through a "microorganism-metabolism-sika deer antler growth axis".Research finds that the guar gum and the xylo-oligosaccharide not only enhance the ability of rumen microorganisms to degrade plant polysaccharides, but also promote the utilization of polysaccharides from the host source, can promote the growth of the sika deer antler, and have a significant promoting effect on the proliferation and mineralization process of the sika deer antler.Adding the guar gum and / or the xylo-oligosaccharide to a total mixed ration of the sika deer with a ratio of concentrate to roughage of 55:45 can significantly increase the weight of the sika deer antler.The application provides an effective scheme for increasing the yield of the sika deer antler, and has important conversion potential in the prevention and treatment of osteoporosis, bone regeneration medicine and the development of functional feed.

Description

Technical Field

[0001] This invention belongs to the field of animal husbandry technology, specifically relating to the application of guar gum and xylooligosaccharides in promoting the growth of sika deer antlers. Background Technology

[0002] Deer antlers are paired bony appendages of the skull unique to cervids, possessing distinctive biological characteristics. Their growth rate is extremely rapid, reaching approximately 1.7 cm / day, even exceeding the growth rate of some cancerous tissues. This unique phenomenon makes deer antlers an important natural model for studying rapid bone growth. The gastrointestinal microbiota plays a broad regulatory role in the physiological functions of the host throughout its life cycle, particularly significantly affecting bone metabolism. Exploring the molecular mechanisms influencing deer antler growth may have important implications for the prevention and treatment of osteoporosis.

[0003] The growth process of deer antlers is highly dependent on nutrient supply. In ruminants, short-chain fatty acids (SCFAs) produced in the gastrointestinal tract (GIT), including acetic acid, propionic acid and butyric acid, are key to energy supply and physiological activities. However, their specific mechanisms of action in the development of deer antlers and bones remain unclear.

[0004] From a histological perspective, deer antlers can be divided into the mesenchymal layer (RM), cartilage layer (CA), and ossification layer (MC). Different layers are enriched with genes related to cell proliferation, angiogenesis, cartilage differentiation, and ossification, respectively. The expression regulation of these genes directly affects antler growth efficiency. However, significant research gaps and application bottlenecks remain in the current technological field: First, while existing research confirms that polysaccharides and oligosaccharides affect the Gibberellin (GIT) microbiota and SCFAs (superficial fibroblast growth factor) generation, it is impossible to determine which polysaccharides or oligosaccharides can efficiently adapt to the GIT microbial characteristics of sika deer to achieve targeted promotion of antler growth. Second, the specific mechanisms by which polysaccharides and oligosaccharides regulate antler growth are insufficiently understood, and their regulatory patterns on gene expression and signaling pathways in different antler layers are not clearly defined. Third, a well-defined and efficiently implementable carbohydrate-based dietary regulation technology for sika deer antler growth has not yet been developed in the field, making it difficult to directly guide antler production increases in large-scale sika deer farming. Therefore, there is an urgent need to develop a technical solution that can accurately match the microbial characteristics of sika deer GIT, clarify the regulatory mechanism, and be reusable, in order to meet the actual needs of increasing the production and quality of deer antlers. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of guar gum and xylooligosaccharides in promoting the growth of sika deer antlers.

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

[0007] In a first aspect, the present invention provides a feed additive that promotes the growth of sika deer antlers, the feed additive comprising guar gum and / or xylooligosaccharides.

[0008] The feed additive consists of guar gum and xylooligosaccharides, with a mass ratio of guar gum to xylooligosaccharides of 1:1.

[0009] In a second aspect, the present invention provides the use of the above-mentioned feed additive in any one of the following (1)-(3):

[0010] (1) Prepare products that promote the growth of deer antlers;

[0011] (2) Prepare products that promote bone growth;

[0012] (3) Prepare products that regulate the gastrointestinal microbiota.

[0013] The regulation of the gastrointestinal microbiota specifically manifests as the promotion of short-chain fatty acid production.

[0014] The short-chain fatty acids include acetic acid and butyric acid.

[0015] In a third aspect, the present invention provides a method for promoting the growth of sika deer antlers, comprising the following steps:

[0016] The above-mentioned feed additives are mixed with the total mixed ration (TMR) and fed to sika deer, with the mass ratio of the feed additives to the TMR being (3-6):100.

[0017] The total mixed ration is prepared by mixing concentrate and roughage at a dry matter ratio of 55:45.

[0018] The beneficial effects of this invention are:

[0019] This invention reveals for the first time that guar gum and xylooligosaccharides synergistically promote antler growth through a "microbe-metabolism-antler growth axis." Studies have found that guar gum and xylooligosaccharides not only enhance the ability of rumen microorganisms to degrade plant polysaccharides but also promote the utilization of host-derived polysaccharides, thus promoting antler growth and significantly enhancing antler proliferation and mineralization. Adding guar gum and / or xylooligosaccharides to a 55:45 concentrate-to-roughage total mixed diet for sika deer significantly increased antler weight. This invention provides an effective solution for increasing antler production in sika deer and offers a theoretical basis for developing intervention strategies for bone metabolic diseases based on prebiotics and microbial regulation, particularly showing significant translational potential in osteoporosis prevention and treatment, bone regeneration medicine, and the development of functional feeds. Attached Figure Description

[0020] Figure 1The effects of XOS and GG on in vitro fermentation in the rumen of sika deer. (A) Comparison of rumen fluid fermentation parameters after 24 h of in vitro fermentation. (B) Comparison of sucrase and xylanase activities in rumen fluid. P <0.05, ** P <0.01.

[0021] Figure 2 The effects of XOS and GG on the composition of microbial fermentation in the rumen of sika deer. (A) Microbial composition at the phylum level and (B) genus level.

[0022] Figure 3 The effects of XOS and GG on the rumen microbiota of sika deer in vitro fermentation. (A) Genera showing significant changes in XOS, GG, INU, and PEC groups compared to the CON group. (B) CCA indicating the correlation between the microbial community and rumen fermentation parameters. Circles represent CON (light gray), XOS (cyan), GG (brown), INU (coral), and PEC (light blue), respectively.

[0023] Figure 4 Functional characteristics of the rumen microbiota of sika deer. (A) Bubble plot showing the differential abundance of the CAZyme family among the CON, XOS, and GG groups. (B) Comparison of rumen fermentation parameters among the CON, XOS, and GG groups. * P <0.05, ** P <0.01.

[0024] Figure 5 Comparison of serum TP, HDL-C and TG levels in the CON, XOS and GG groups.

[0025] Figure 6 Serum bile acid levels were analyzed in the CON, XOS, and GG groups. (A) PLS-DA showed the differences in serum bile acid profiles among the three groups, and the pie chart showed the relative proportions of primary, secondary, conjugated, and unconjugated bile acids. (B) Total serum bile acid levels and significantly different bile acids among the three groups.

[0026] Figure 7 Serum fatty acid and growth factor levels were analyzed in the CON, XOS, and GG groups. (A) Comparison of serum free fatty acid concentrations among the three groups. (B) Comparison of serum growth factor levels among the three groups.

[0027] Figure 8 Safranin O–Fix Green staining was performed on deer antler tissues from the CON, XOS, and GG groups.

[0028] Figure 9 Volcano plots of differentially expressed genes in RM, CA, and MC.

[0029] Figure 10Transcriptomic analysis of deer antler development. Comparison of RM(A) and MC(B) gene expression.

[0030] Figure 11 Analysis of mesenchymal cells (RMCs) treated with sodium taurodeoxychenobilate (THDCA). (A) ALP activity and staining area in RMCs after THDCA treatment. (B) Upregulation of gene expression and (C) protein expression (THDCA-treated group). Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0033] The oligosaccharides and polysaccharides used in the examples are all commercially available products. The CAS number of the guar gum used is 39421-75-5, and the CAS number of the xylooligosaccharide used is 87-99-0.

[0034] The animal experiments in this example comply with the requirements of the Animal Ethics Committee of Jilin Agricultural University's "Guidelines for Animal Research" (Approval No.: 20210314002).

[0035] Example 1: Screening of oligosaccharides and polysaccharides affecting in vitro rumen fermentation

[0036] To investigate the effects of oligosaccharides and polysaccharides on rumen fermentation, the effects of oligosaccharides (AOS), fructooligosaccharides (FOS), and xylooligosaccharides (XOS) and polysaccharides (GG), pectin (PEC), and inulin (INU) on rumen fluid fermentation in sika deer during the antler-growing period were first studied using in vitro fermentation methods.

[0037] The in vitro fermentation steps are as follows:

[0038] A gas production measurement system (RFS, ANKOM, USA) was used. Specifically, 120 mL of premixed solution (80 mL buffer and 40 mL rumen fluid) was added to each fermentation flask, CO2 gas was introduced for 30 s, and the flask was immediately capped and placed in an air bath incubator (HNY-211B, Honour, Tianjin). Gas production was recorded using GPM software (ANKOM, USA). Corn silage was used as the fermentation substrate. The control group (CON) received only the substrate, while the other treatment groups received 3% DM of oligosaccharides or polysaccharides in their substrates, including xylo-oligosaccharides (XOS, 95% purity), fructo-oligosaccharides (FOS, 95% purity), alginate-oligosaccharides (AOS, 100% purity), inulin (INU, 90% purity), guar gum (GG, 99.7% purity), and pectin (PEC, 74% purity). After 24 h of fermentation, the fermentation flasks were removed and immediately placed in ice water to terminate fermentation. Three replicates were performed at each time point (one replicate per fermentation flask). The pH of the fermentation broth was measured using a pH meter (MP511, Sanxin, Shanghai). The collected fermentation broth was then immediately frozen in liquid nitrogen and stored at -80 °C for further analysis.

[0039] Experimental results showed that the concentrations of TSCFAs, acetic acid, and butyric acid were significantly increased in the XOS, GG, and INU groups. P <0.05, Fig. 1A), and significantly reduced pH ( P <0.05%, isovaleric acid and valeric acid concentrations were significantly increased only in the XOS and GG groups ( P <0.05, Figure 1 A). However, there were no significant differences in gas production, lactic acid (a precursor to butyric acid), and propionic acid concentration among the groups. P >0.05). Butyrate can be generated from glutamic acid and lysine, therefore it is speculated that the elevated butyrate level may mainly originate from amino acid degradation rather than lactate metabolism. Sucrase activity was significantly higher in the XOS and GG groups than in other groups ( P <0.05, Figure 1 B), while xylanase activity increased in the PEC group. Cellulase and α-amylase activities did not differ significantly ( PThe value >0.05 indicates that different treatments have varying effects on the activity of carbohydrate-degrading enzymes. These results demonstrate that XOS and GG can effectively improve the fermentation efficiency and SCFA yield of rumen fluid in vitro.

[0040] Further 16S rRNA gene sequencing of rumen fluid yielded 434,524 high-quality 16S rRNA gene sequences (mean = 28,968, range 27,373-29,863), which were classified into 1,339 ASVs belonging to 11 phyla and 115 genera. Bacteroidota and Bacillota were the dominant bacterial phyla. Figure 2 A), Prevotella Rikenellaceae RC9 Succiniclasticum It is the dominant bacterial genus ( Figure 2 B).

[0041] Based on the Bray-Curtis dissimilarity matrix ( P = 0.04), unweighted UniFrac matrix ( P = 0.04, Figure 3 Principal coordinate analysis (PCoA) showed that oligosaccharides and polysaccharides significantly influenced the rumen microbial community structure. Canonical correlation analysis (CCA) further indicated that these microorganisms were positively correlated with acetic acid, butyric acid, TSCFAs, and sucrase activity. Figure 3 B). XOS and GG play a central role in the polysaccharide and amino acid metabolism of ruminants, promoting the production of acetic acid and butyric acid. These results indicate that XOS and GG can optimize rumen fermentation by reshaping the microbial community structure.

[0042] Example 2: Investigation on the effects of xylooligosaccharides and guar gum on the growth of sika deer antlers

[0043] This experiment used 18 healthy male sika deer, aged 4 years and raised in captivity (average weight: 89.3 ± 1.7 kg). Each deer was housed in a separate enclosure and fed twice daily, at 7:00 AM and 4:00 PM, with free access to water. The animals were randomly divided into three groups: CON, XOS, and GG, with six animals in each group. The three groups were fed a total mixed ration (concentrate to roughage ratio of 55:45, dry matter basis, Table 1) supplemented with 0 g / kg, 30 g / kg XOS, and 30 g / kg GG (all dry matter basis), respectively. The experiment lasted 7 weeks, with the first week being the acclimatization period and the following 6 weeks being the formal experimental period. Daily feed intake and uneaten feed were recorded.

[0044] Table 1. Composition and nutrient levels of basal diets (dry matter basis)

[0045]

[0046] Each kilogram of premix contains: 200 g NaHCO3, 75 g Ca, 20 g P, 600 mg Mn, 680 mg Fe, 960 mg Zn, 300 mg Cu, 140,000 IU vitamin A, 2,000 IU vitamin D3, and 700 IU vitamin E.

[0047] On the last day of the feeding trial, to analyze the effects of GG and XOS on rumen microbial function and metabolism, the total genome of sika deer rumen fluid, feces, and in vitro rumen fermentation broth was extracted using the bead milling method and shotgun sequencing (194 Gb) was performed. A non-redundant rumen gene set of 21.4 million genes was constructed, significantly higher than that of roe deer (13.6 million) and water deer (7.6 million) as previously reported. These results indicate that a highly adaptive and diverse microbial community exists in the rumen of sika deer during the antler growth period.

[0048] Thirty-nine CAZymes family members were identified in the rumen of sika deer. Gas chromatography (7890B, Agilent, UK) was used to detect the concentrations of short-chain fatty acids (SCFAs) in in vitro rumen fermentation broth, rumen fluid, and feces. Ammonia nitrogen in rumen fluid was determined using a UV-Vis spectrophotometer (UV-1201, Shimadzu, Kyoto, Japan). Lactic acid concentration and the activities of α-amylase, cellulase, and sucrase were detected using a kit (Jiancheng Bioengineering Institute, Nanjing, China). Xylanase activity in the fermentation broth was detected using enzyme-linked immunosorbent assay (ELISA) and a kit. The activity of hemicellulase (GH43, PL11, CE1), cellulase (GH3, CBM6), amylase (GH13, CBM48), and pectinase (PL1) revealed their ability to efficiently degrade polysaccharides derived from woody plants. Furthermore, the number of rumen host glycanase family (GH20, GH156, CE9) genes in the GG and XOS groups was significantly higher than that in the CON group. P <0.05), among which the levels of cellulase, hemicellulase, pectinase, and amylase family (such as GH1, GH48, GH130, and GH63) in the GG group were significantly higher than those in the CON group ( P <0.05, Figure 4 A). The increase in total SCFAs, acetic acid, butyric acid, and branched-chain fatty acid levels in the GG group was consistent with ( P <0.05, Figure 4(B) The results showed that GG mainly improves fermentation efficiency and energy uptake by enhancing the degradation of carbohydrates and amino acids into pyruvate; while XOS mainly promotes microbial growth and community stability by enhancing the fermentation function of the core microorganism.

[0049] To investigate how changes in the rumen and fecal microbiota exert a cascade effect on host metabolism, a fully automated biochemical analyzer (Vitalab Selectra E, Vitalab, Dieren, Netherlands) was used to detect total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglyceride levels in serum samples. The results showed that HDL-C levels in the GG and XOS groups were significantly higher than in the CON group, while total protein (TP) and triglyceride (TG) levels in the GG group were significantly higher than in the CON and XOS groups. Figure 5 The remaining indicators showed no significant differences. These results indicate that GG and XOS significantly affect lipid metabolism in sika deer and are associated with SCFAs production.

[0050] Secondly, an LC-ESI-MS / MS system (UHPLC, ExionLC™ AD; MS, Applied Biosystems 6500 Triple Quadrupole) equipped with a Waters ACQUITY UPLC HSS T3 C18 column (100 mm × 2.1 mm, 1.8 μm) was used for the detection of bile acids. The concentrations of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transgenic growth factor β1 (TGF-β1), fibroblast growth factor 2 (FGF-2), and bone morphogenetic protein-2 (BMP-2) in serum were determined using a kit (Jiancheng Bioengineering Institute, Nanjing, China). The results showed a significant change in the composition of serum bile acids, with increased levels of total bile acids and primary bile acids, and decreased levels of secondary bile acids. Figure 6 A). Further analysis revealed that, compared with the CON and XOS groups, the concentrations of chenodeoxycholic acid, cholic acid, deoxycholic acid, porcine deoxycholic acid, isochenodeoxycholic acid, sodium taurodeoxycholate (THDCA), 23-deoxycholic acid, 7-ketodeoxycholic acid, and 7-ketolithocholic acid were significantly increased in the GG group. Figure 6 B). These results indicate that GG may enhance bile acid deconjugation and alter enterohepatic circulation through enzymes derived from gut microbiota. The levels of oleic acid, α-linolenic acid, palmitic acid, myristic acid, and stearic acid in the GG and XOS groups were significantly lower than those in the CON group. Figure 7 A). Furthermore, the levels of multiple serum growth factors were significantly increased in the GG group ( Figure 7 B), including insulin-like growth factor-1 (IGF-1), transforming growth factor β1 (TGF-β1), platelet-derived growth factor (PDGF), fibroblast growth factor 2 (FGF2), and bone morphogenetic protein 2 (BMP2). These growth factors synergistically regulate osteoblast proliferation, matrix deposition, and angiogenesis, processes that play a crucial role in the rapid growth of deer antlers. The combined effect of elevated bile acids and growth factors suggests that GG may promote antler growth by integrating metabolic and signaling pathway networks.

[0051] Furthermore, deer antler tissue was fixed in 4% paraformaldehyde solution and decalcified in a standard decalcification solution for 4 weeks. Undecalcified and decalcified tissues were cut into 4 μm and 5 μm sections, respectively. Hematoxylin-eosin staining and safranin-fast green staining were performed, and all stained sections were observed and imaged under an optical microscope (Olympus CX31, Japan) for histological analysis of deer antler tissue. The staining results showed that the GG group significantly promoted bone matrix deposition and trabecular bone structure reorganization, revealing a higher degree of ossification, while the XOS group showed relatively weaker ossification. Figure 8 ).

[0052] Differentially expressed genes (DEGs) were analyzed using DESeq2 (v1.42.1), and Metascape was used to perform gene ontology (GO) functional annotation and KEGG pathway enrichment analysis on the selected DEGs. The results showed that the GG group had the highest number of differentially expressed genes, particularly concentrated in RM and MC. Figure 9 The mesenchymal stem cell (RM) is the initial growth region of antler, rich in highly proliferative mesenchymal stem cells and progenitor cells, which determine the potential for subsequent cartilage and bone formation; while the cellular stem cell (MC) is involved in cell differentiation, mineralization, and bone remodeling, and has extremely high metabolic and signaling requirements. These results indicate that glycogen (GG) has a significant promoting effect on the proliferation and mineralization processes of antler.

[0053] KEGG enrichment analysis results showed that the GG group had fewer genes in the PI3K-Akt pathway in RM ( PIK3CA and AKT1 () Figure 10 A) and the Wnt and TGF-β pathways in MC Cyclin D1 , TGF-β1 and SMAD3 Gene expression ( Figure 10 B) were significantly higher than those in the CON group. These results indicate that GG supports rapid growth and ossification of antlers by promoting angiogenesis and osteogenic processes.

[0054] The above analysis of bile acids revealed that the concentration of sodium tauride-deoxychenobilate (THDCA) was significantly higher in the GG and XOS groups compared to the CON group. To further analyze the mechanism of action of GG and XOS on the rapid growth and ossification of deer antler, deer antler mesenchymal cells (RMCs) passaged to the fourth generation were treated with different concentrations of sodium tauride-deoxychenobilate (THDCA, 100 μM and 500 μM). Cells cultured in complete osteogenic induction medium served as the control group. The osteogenic induction medium consisted of DMEM, 10% fetal bovine serum (FBS), 0.1 μM dexamethasone, 0.2 mM vitamin C, and 10 mM sodium β-glycerophosphate. After 7 days of culture, alkaline phosphatase (ALP) staining was performed using a kit (Beyotime, Shanghai, China) to assess the osteogenic differentiation level. Simultaneously, ALP activity was measured using a microplate reader (Synergy H1, BIO-TEK, USA) at a wavelength of 405 nm. At 21 days of culture, Alizarin Red S (ARS) staining was performed to detect the formation of mineralized nodules. For quantitative analysis, the bound dye was dissolved in 10% acetic acid, and the absorbance was measured at 405 nm using a microplate reader (SynergyH1, BIO-TEK, USA). At the same time point, total RNA and protein were extracted from the cells, and the mRNA and protein expression levels of RUNX2 and osteocalcin (OCN) were detected by real-time reverse transcription quantitative PCR and Western blotting. WGCNA analysis showed that THDCA significantly increased the alkaline phosphatase (ALP) content and enzyme activity in mesenchymal cells (RMCs). After 21 days of induction, Alizarin Red S staining results showed that all THDCA-treated groups formed obvious orange-red mineralized nodules, which was significantly better than the control group. Figure 11 A). Meanwhile, the expression levels of key osteogenic transcription factors RUNX2 and OCN were significantly higher in the THDCA-treated group than in the control group ( Figure 11 B- Figure 11 (C), indicating that THDCA can promote osteogenic differentiation of RMCs.

[0055] Based on the above results, it can be seen that both XOS and GG can affect the rapid growth and ossification of deer antlers to varying degrees. To further investigate the effects of GG, XOS, and the combination of GG and XOS on the weight of deer antlers, an XOS+GG group was added to the CON, XOS, and GG groups. The XOS+GG group received a total mixed ration (concentrate to roughage ratio of 55:45, based on dry matter, Table 1) with 30 g / kg of feed additive (XOS and GG mixed at a mass ratio of 1:1, based on dry matter). Other experimental conditions were the same as the other treatment groups. On the last day of the feeding experiment, deer antlers from the CON, XOS, GG, and XOS+GG groups were collected and weighed 3 hours after morning feeding. The statistical results are shown in Table 2.

[0056] Table 2: Statistics on Antler Growth

[0057]

[0058] Experimental results showed that adding GG and XOS to the diet of sika deer both increased the weight of antlers, with the combination of GG and XOS showing the most significant effect.

[0059] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted.

[0060] Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A method for promoting the growth of antlers in male sika deer during the antler-growing period, characterized in that, Includes the following steps: (1) Guar gum and xylooligosaccharides are mixed at a mass ratio of 1:1 to obtain a feed additive; (2) Mix the feed additives with the total mixed ration at a mass ratio of (3-6):100 and feed them to male sika deer in the antler growth period.

2. The method according to claim 1, characterized in that, In step (2), the total mixed ration is obtained by mixing concentrate and roughage at a dry matter mass ratio of 55:45.

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