Application of guar gum and xylooligosaccharide in promoting growth of cornu cervi pantotrichum of sika deer

Abstract

The invention discloses application of guar gum and xylooligosaccharide in promoting growth of cornu cervi pantotrichum of sika deer, and belongs to the technical field of animal breeding. The invention reveals that guar gum and xylooligosaccharide synergistically promote the growth of pilose antler through'microorganism-metabolism-pilose antler growth axis' for the first time, and researches find that guar gum and xylooligosaccharide not only enhance the ability of rumen microorganisms to degrade plant polysaccharides, but also promote the utilization of host-derived polysaccharides, can promote the growth of pilose antler, and can promote the growth of pilose antler. The obvious promoting effect is achieved on the pilose antler proliferation and mineralization process. The guar gum and / or xylooligosaccharide are / is added into the sika deer total mixed ration with the concentrate-to-coarse ratio of 55: 45 to feed sika deer, so that the weight of pilose antler is remarkably increased. The invention provides an effective scheme for increasing the yield of the cornu cervi pantotrichum of the sika deer, and particularly has important transformation potential in the aspects of osteoporosis prevention and treatment, bone regeneration medicine and functional feed development.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal breeding, and particularly relates to application of guar gum and xylo-oligosaccharide in promoting growth of sika deer antler. BACKGROUND

[0002] Antler is a paired cranial bony appendage unique to Cervidae, with unique biological characteristics. The growth rate of antler is extremely fast, which can reach about 1.7 cm / day, even exceeding the growth rate of some cancer tissues. This special phenomenon makes antler an important natural model for studying rapid bone growth. The gastrointestinal microbiota has a wide regulatory effect on the physiological functions of the host life cycle, especially on bone metabolism. Exploring the molecular mechanism affecting the growth of antler may have important significance for the prevention and treatment of osteoporosis.

[0003] The growth process of antler has extremely high dependence 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 the key to energy supply and physiological activities of the body, however, the specific mechanism of action in the process of antler and bone development is still unclear.

[0004] From the histological characteristics of antler growth, antler can be divided into mesenchymal layer (RM), cartilage layer (CA) and ossification layer (MC), and different layers are rich in genes related to cell proliferation, angiogenesis, cartilage differentiation and ossification, and the expression regulation of these genes directly affects the growth efficiency of antler. However, there are still significant research gaps and application bottlenecks in the current technical field: first, although existing researches have confirmed that polysaccharides and oligosaccharides have an impact on the GIT microbiota and the production of SCFAs, it is still unclear which polysaccharides or oligosaccharides can efficiently adapt to the GIT microbial characteristics of sika deer to achieve directional promotion of antler growth; second, the specific mechanism of polysaccharides and oligosaccharides in regulating antler growth is not clear, and the regulation rules of gene expression and signal pathways in different layers of antler are also not clear; third, there is still no carbohydrate diet regulation technology for sika deer antler growth with clear mechanism and high efficiency in the current field, which is difficult to directly guide the antler yield increase practice in large-scale sika deer breeding. Therefore, it is urgent to develop a technical solution that can accurately match the GIT microbial characteristics of sika deer, has a clear regulation mechanism and can be repeatedly applied to meet the actual needs of antler yield increase and quality improvement. SUMMARY

[0005] In view of the above prior art, the purpose of the present application is to provide application of guar gum and xylo-oligosaccharide in promoting growth of sika deer antler.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present application is as follows: In a first aspect of the present application, a feed additive for promoting the growth of sika deer antler is provided, the feed additive comprising guar gum and / or xylo-oligosaccharide.

[0007] The feed additive consists of guar gum and xylo-oligosaccharide, and the mass ratio of the guar gum and the xylo-oligosaccharide is 1:1.

[0008] In a second aspect of the present application, the above-mentioned feed additive is applied in any one of the following (1)-(3): (1) preparing a product for promoting the growth of antler; (2) preparing a product for promoting the growth of bone; (3) preparing a product for regulating the gastrointestinal microbiota.

[0009] The regulation of the gastrointestinal microbiota specifically manifests as promotion of the generation of short-chain fatty acids.

[0010] The short-chain fatty acids include acetic acid and butyric acid.

[0011] In a third aspect of the present application, a method for promoting the growth of sika deer antler is provided, comprising the following steps: The above-mentioned feed additive is mixed with a total mixed ration for feeding sika deer, and the mass ratio of the feed additive to the total mixed ration is (3-6):100.

[0012] The total mixed ration is obtained by mixing concentrate and roughage at a dry matter mass ratio of 55:45.

[0013] Advantages of the present application: The present application first discloses that guar gum and xylo-oligosaccharide promote the growth of antler through a "microorganism-metabolism-antler growth axis". Research has found that guar gum and xylo-oligosaccharide not only enhance the ability of rumen microorganisms to degrade plant polysaccharides, but also promote the utilization of polysaccharides from the host, and can promote the growth of antler and significantly promote the proliferation and mineralization process of antler. Adding guar gum and / or xylo-oligosaccharide to the total mixed ration of sika deer with a concentrate to roughage ratio of 55:45 can significantly increase the weight of sika deer antler. The present application provides an effective solution for increasing the yield of sika deer antler and provides a theoretical basis for developing bone metabolism disease intervention strategies based on prebiotics and microbial regulation, which has important transformation potential in the prevention and treatment of osteoporosis, bone regeneration medicine and functional feed development. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Effects of XOS and GG on in vitro rumen fermentation of sika deer. (A) Comparison of fermentation parameters of rumen fluid after 24 h of in vitro fermentation. (B) Comparison of the activities of sucrase and xylanase in rumen fluid, P <0.05, ** P<0.01.

[0015] Figure 2 Effects of XOS and GG on the rumen microbial composition of sika deer in vitro fermentation. (A) Microbial composition at the phylum level and (B) at the genus level.

[0016] Figure 3 Effects of XOS and GG on the rumen microbiota of sika deer in vitro fermentation. (A) Genus with significant changes in XOS, GG, INU and PEC groups compared with CON group. (B) CCA indicated the correlation of rumen microbial community with rumen fermentation parameters. Circles represent CON (light gray), XOS (cyan), GG (brown), INU (coral) and PEC (light blue), respectively.

[0017] Figure 4 Functional characteristics of rumen microbiota of sika deer. (A) Bubble chart indicated the differentially abundant CAZyme families among CON, XOS and GG groups. (B) Comparison of rumen fermentation parameters among CON, XOS and GG groups. P <0.05, ** P <0.01.

[0018] Figure 5 Comparison of serum TP, HDL-C and TG levels among CON, XOS and GG groups.

[0019] Figure 6 Analysis of serum bile acid levels in CON, XOS and GG groups. (A) PLS-DA indicated the differences in serum bile acid profiles among the three groups, and pie charts indicated the relative proportions of primary, secondary, conjugated and non-conjugated bile acids. (B) Comparison of total serum bile acid levels and significantly different bile acids among the three groups.

[0020] Figure 7 Analysis of serum fatty acid and growth factor levels in 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.

[0021] Figure 8 H&E staining of Cervi Cornu Pantotrichum tissues in CON, XOS and GG groups.

[0022] Figure 9 Volcano plot of differentially expressed genes in RM, CA and MC.

[0023] Figure 10 Transcriptomic analysis of Cervi Cornu Pantotrichum development stages. Comparison of gene expression in RM (A) and MC (B).

[0024] Figure 11Analysis 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

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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).

[0029] Example 1: Screening of oligosaccharides and polysaccharides affecting in vitro rumen fermentation 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.

[0030] The in vitro fermentation steps are as follows: 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.

[0031] 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.

[0032] 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 genus of bacteria ( Figure 2 B).

[0033] 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.

[0034] Example 2: Investigation on the effects of xylooligosaccharides and guar gum on the growth of sika deer antlers 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.

[0035] Table 1. Composition and nutrient levels of basal diets (dry matter basis) 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 ).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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. Table 2: Statistics on Antler Growth 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.

[0045] 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. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. A feed additive for promoting the growth of sika deer velvet, characterized by comprising a mixture of the following components: The feed additive comprises guar gum and / or xylo-oligosaccharide. ​ 2. The feed supplement according to claim 1, characterized in that, The feed additive consists of guar gum and xylo-oligosaccharide, and the mass ratio of the guar gum and the xylo-oligosaccharide is 1:

1.

3. The feed additive of claim 1 or 2 is used in any one of the following (1)-(3): (1) to prepare a product for promoting the growth of pilose antler; (2) to prepare a product for promoting the growth of bone; (3) to prepare a product for regulating the gastrointestinal microbiota.

4. Use according to claim 3, characterized in that, The regulation of the gastrointestinal microbiota specifically manifests as promotion of the generation of short-chain fatty acids.

5. Use according to claim 4, characterized in that, The short-chain fatty acids include acetic acid and butyric acid.

6. A method for promoting the growth of sika deer velvet, characterized by, The method comprises the following steps: The feed additive of claim 1 or 2 is mixed with a total mixed ration to feed the sika deer, and the mass ratio of the feed additive to the total mixed ration is (3-6):

100.

7. The method of claim 6, wherein, The total mixed ration is obtained by mixing concentrate and roughage at a dry matter mass ratio of 55:45.

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