核桃青皮提取物对绵羊瘤胃体外发酵及微生物区系的影响

doi:10.16431/j.cnki.1671-7236.2024.03.008

摘要/Abstract

摘要： 【目的】研究核桃青皮提取物对绵羊瘤胃体外发酵参数及微生物区系的影响,以阐明其在调控瘤胃发酵中的作用。【方法】以精粗比为65∶35的饲粮为底物,分别添加0、0.125%、0.25%、0.50%和1.00%(底物质量)的乙酸乙酯(Ea)、水(Dw)及50%乙醇(Ha)核桃青皮提取物,体外发酵24 h。发酵期间记录产气量,发酵结束后测定发酵参数、底物降解率、乳酸(LA)浓度及相关酶活性;基于16S rDNA V4区高通量测序分析绵羊瘤胃细菌区系的变化。【结果】与对照组相比,Ea组pH、干物质降解率(DMD)、乙丙比、6-磷酸果糖激酶(PFK)、己糖激酶(HK)及丙酮酸激酶(PK)活性均极显著升高(P＜0.01),24 h累积产气量(GP_{24 h})、原虫数、氨态氮(NH₃-N)及乙酸、丙酸、戊酸、总挥发性脂肪酸(TVFA)和LA浓度极显著或显著降低(P＜0.01;P＜0.05);Dw组pH、GP_{24 h}、中性洗涤纤维降解率(NDFD)、丙酸、异丁酸、戊酸、LA浓度及PFK、HK和PK活性极显著或显著降低(P＜0.01;P＜0.05),乙丙比极显著升高(P＜0.01);Ha组GP_{24 h}、DMD、NDFD、异丁酸、丁酸、异戊酸浓度及乙丙比极显著或显著降低(P＜0.01;P＜0.05),原虫数、NH₃-N及丙酸浓度均极显著或显著升高(P＜0.01;P＜0.05)。16S rDNA测序表明,与对照组相比,Ea组Alpha多样性指数、厚壁菌门、拟杆菌门及肠群理研菌属RC9相对丰度极显著或显著升高(P＜0.01;P＜0.05),变形菌门、琥珀酸弧菌属和解琥珀酸菌属相对丰度极显著或显著降低(P＜0.01;P＜0.05);Dw组Shannon指数、拟杆菌门、肠群理研菌属RC9及未培养瘤胃细菌相对丰度极显著或显著降低(P＜0.01;P＜0.05);Ha组Alpha多样性指数及厚壁菌门相对丰度均极显著降低(P＜0.01),琥珀酸弧菌属、普雷沃氏菌属及解琥珀酸菌属相对丰度显著或极显著升高(P＜0.05;P＜0.01)。【结论】在饲粮中添加核桃青皮乙酸乙酯提取物后可有效改善绵羊瘤胃发酵,添加剂量以底物质量的0.25%效果最好。

关键词: 核桃青皮提取物; 绵羊; 体外发酵; 微生物区系

Abstract: 【Objective】 This study was conducted to examine the effect of walnut green husk (WGH) extracts on in vitro rumen fermentation and microbiota community in sheep,in order to elucidate its role in regulating rumen fermentation.【Method】 The WGH extracts obtained using ethyl acetate (Ea),deionized water (Dw),and 50% hydroalcohol (Ha) were added at concentrations of 0,0.125%,0.25%,0.50%,and 1.00% (weight of substrate) and fermented in vitro for 24 h using a high concentrate ration (the ratio of concentrate/roughage was 65∶35) as substrate.Gas production (GP) was recorded during fermentation,and at the end of the fermentation,the fermentation parameters,substrate degradation rate,lactate (LA) concentration and related enzyme activities were analyzed.Additionally,the changes in rumen bacterial community were assessed using high-throughput sequencing of the 16S rDNA V4 region.【Result】 Compared with control group,pH,dry matter degradation rate (DMD),acetate/propiontate (A/P ratio),and the activities of 6-phosphofructokinase (PFK),hexokinase (HK) and pyruvate kinase (PK) in Ea group were extremely significantly increased (P＜0.01),the 24-hour cumulative gas production (GP_{24 h}),protozoa number,and the concentrations of ammonia nitrogen (NH₃-N),acetate,propiontate,valerate,total volatile fatty acids (TVFA) and LA were extremely significantly or significantly desreased (P＜0.01 or P＜0.05).Compared with control group,pH,GP_{24 h},neutral detergent fiber degradation rate (NDFD),the levels of propiontate,isobutyrate valerate,and LA,and the activities of PFK,HK and PK in Dw group were extremely significant or significantly decreased (P＜0.01 or P＜0.05),while the A/P ratio exhibited an extremely significant increase (P＜0.01).As for Ha group,GP_{24 h},DMD,NDFD,the levels of isobutyric acid,butyrate and isovalerate,and A/P ratio were extremely significantly or significantly decreased reduced (P＜0.01 or P＜0.05),the protozoa count and the levels of NH₃-N and propiontate were extremely significantly or significantly increased (P＜0.01 or P＜0.05).16S rDNA sequencing showed that compared with control group,Alpha diversity index and the relative abundances of Firmicutes,Bacteroidotes and Rikenellaceae_RC9_gut_group in Ea group were extremely significantly or significantly increased (P＜0.01 or P＜0.05),whereas the relative abundances of Proteobacteria,Succinivibrio and Succiniclasticum were exhibited an extremely significant or significant decrease (P＜0.01 or P＜0.05).The Shannon index and the relative abundances of Bacteroidotes,Rikenellaceae_RC9_gut_group and uncultured rumen bacterium in Dw group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05).Alpha diversity index and the abundance of Firmicutes in Ha group were extremely significantly deceased (P＜0.01),and the relative abundances of Succinivibrio,Prevotella and Succiniclasticum were significantly or extremely significantly increased (P＜0.05 or P＜0.01).【Conclusion】 The addition of ethyl acetate extract of walnut green husk in the diet could effectively improve rumen fermentation in sheep,with the best results observed when added at a dosage of 0.25% based on substrate weight.

Key words: walnut green husk extracts; sheep; in vitro fermentation; microbiota community

中图分类号:

S816.6

张萧, 李璇玥, 王蕾, 方美烟, 田聪聪, 狄明岳, 陈勇. 核桃青皮提取物对绵羊瘤胃体外发酵及微生物区系的影响[J]. 中国畜牧兽医, 2024, 51(3): 967-977.

ZHANG Xiao, LI Xuanyue, WANG Lei, FANG Meiyan, TIAN Congcong, DI Mingyue, CHEN Yong. Effects of Walnut Green Husk Extracts on in vitro Rumen Fermentation and Microbiota Community in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(3): 967-977.

参考文献

[1] 张涛,牟英玉,亓王盼,等.亚急性瘤胃酸中毒耐受性不同的奶牛瘤胃上皮形态及功能差异研究[J].草业学报, 2023, 32(2):131-139. ZHANG T, MU Y Y, QI W P, et al.Comparison of rumen epithelium morphology and function in dairy cows with differences in susceptibility for subacute ruminal acidosis[J].Acta Prataculturae Sinica, 2023, 32(2):131-139.(in Chinese)
[2] ZHANG T, MU Y, ZHANG R, et al.Responsive changes of rumen microbiome and metabolome in dairy cows with different susceptibility to subacute ruminal acidosis[J].Animal Nutrition, 2022, 8(1):331-340.
[3] 杨洋.有机酸和瘤胃素组合对SARA奶山羊瘤胃发酵和细菌的影响[D].呼和浩特:内蒙古农业大学, 2009. YANG Y.Effect of adding both disodium fumarate and monensin on ruminal fermentation and its bacterial amounts in dairy goats suffered from subacute rumen acidosis[D].Hohhot:Inner Mongolia Agricultural University, 2009.(in Chinese)
[4] 郭永兰,陈功哲.奶牛亚急性瘤胃酸中毒的诊断和防治方法研究[J].特种经济动植物, 2022, 25(10):76-78. GUO Y L, CHEN G Z.Studies on the diagnosis and prevention of subacute rumen acidosis in dairy cows[J].Special Economic Animals and Plants, 2022, 25(10):76-78.(in Chinese)
[5] 安文娟.辣椒素对肉牛亚急性瘤胃酸中毒的缓解作用机理研究[D].泰安:山东农业大学, 2022. AN W J.Mechanistic study on the alleviating effect of capsaicin on subacute ruminal acidosis in beef cattle[D].Tai'an:Shandong Agricultural University, 2022.(in Chinese)
[6] AHMED M G, AL S A A, EL Z S Z, et al.Potential of selected plant extracts to control severe subacute ruminal acidosis in vitro as compared with monensin[J].BMC Veterinary Research, 2022, 18(1):356.
[7] 陈威.核桃青皮提取物预防亚急性瘤胃酸中毒作用的研究[D].乌鲁木齐:新疆农业大学, 2022. CHEN W.Study on the preventive of walnut green husk extract on subacute ruminal acidosis[D] Urumqi:Xinjiang Agricultural University, 2022.(in Chinese)
[8] MENKE K H, STEINGASS H.Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid[J].Animal Research and Development, 1988, 28:7-55.
[9] 冯仰廉.反刍动物营养学[M].北京:科学出版社, 2004. FENG Y L.Ruminant Nutrition[M].Beijing:Science Press, 2004.(in Chinese)
[10] 王加启.反刍动物营养学研究方法[M].北京:现代教育出版社, 2011. WANG J Q.Research Methods in Ruminant Nutrition[M].Beijing:Modern Education Press, 2011.(in Chinese)
[11] 许晓莉.添加大豆磷脂对绵羊瘤胃消化代谢及微生物区系的影响[D].乌鲁木齐:新疆农业大学, 2013. XU X L.Effects of dietary addition of soybean lecithin on ruminal metabolism and microbial diverisity in sheep[D].Urumqi:Xinjiang Agricultural University, 2013.(in Chinese)
[12] 李晴.添加不同组合支链氨基酸对绵羊整体消化代谢及瘤胃微生物多样性的影响[D].乌鲁木齐:新疆农业大学, 2015. LI Q.Effects of different combinations branched-chain amino acid on sheep whole digestion metabolism and rumen microbial diversity[D].Urumqi:Xinjiang Agricultural University, 2015.(in Chinese)
[13] 张丽英.饲料分析及饲料质量检测技术[M].3版.北京:中国农业大学出版社, 2007. ZHANG L Y.Feed Analysis and Feed Quality Testing Techniques[M].3rd ed.Beijing:China Agricultural University Press, 2007.(in Chinese)
[14] 中华人民共和国农业农村部.饲料中酸性洗涤纤维的测定NY/T 1459-2022[S].北京:中国农业出版社, 2022. MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT OF THE PEOPLE'S REPUBLIC OF CHINA.Determination of acid detergent fibers in feed:NY/T 1459-2022[S].Beijing:China Agriculture Press, 2022.(in Chinese)
[15] NURFITRIANI R A, JAYANEGARA A, KUMALASARI N R, et al.Effects of bionanomineral selenium (Bionano-Se) and probiotics inclusion to ration on in vitro rumen fermentation characteristics[J].Journal of Animal&Plant Sciences, 2023, 33(1):67-74.
[16] SALLAM S M A, NASSER M E A, EL W A M, et al.Use of an in vitro rumen gas production technique to evaluate some ruminant feedstuffs[J]. Journal of Applied Sciences Research, 2007, 3(1):34-41.
[17] 王志敬,吴征敏,庄桂锋,等.不同精粗比日粮对雷州山羊体外瘤胃发酵的影响[J].中国畜牧兽医, 2017, 44(8):2303-2310. WANG Z J, WU Z M, ZHUANG G F, et al.Effects of diets with different concentrate to forage ratios on in vitro rumen fermentation of Leizhou goats[J]. China Animal Husbandry&Veterinary Medicine, 2017, 44(8):2303-2310.(in Chinese)
[18] WANG Y, WANG L, WANG Z, et al.Recent advances in research in the rumen bloat of ruminant animals fed high-concentrate diets[J].Frontiers in Veterinary Science, 2023, 10:1142965.
[19] FERME D, BANJAC M, CALSAMIGLIA S, et al.The effects of plant extracts on microbial community structure in a rumen-simulating continuous-culture system as revealed by molecular profiling[J]. Folia Microbiologica, 2004, 49:151-155.
[20] JI X, TONG H, SETTLAGE R, et al.Establishment of a bovine rumen epithelial cell line[J].Journal of Animal Science, 2021, 99(10):skab273.
[21] 包乌日汉,青春,曲永利,等.丝兰提取物与日粮精粗比对体外发酵和甲烷产生量的交互影响[J].饲料工业, 2023, 44(21):32-39. BAO W R H, QING C, QU Y L, et al.Effects of Yucca schidigera extraction in vitro fermentation and methane production in a variety of diets of forage to concentrate ratio[J].Feed Industry.2023, 44(21):32-39.(in Chinese)
[22] SUCU E.In vitro studies on rumen fermentation and methanogenesis of different microalgae and their effects on acidosis in dairy cows[J].Fermentation, 2023, 9(3):229.
[23] HE B, FAN Y, ZHAO X, et al.Lactate transport and metabolism in rumen epithelial cells in SARA condition[J].Italian Journal of Animal Science, 2023, 22(1):239-249.
[24] KACHHADIA L, KATOLE S B, PATEL N.Acidosis in ruminants:Nutritional overview[J].The Pharma Innovation, 2023, 12(1):20-22.
[25] 李颖.猴头菇多糖缓解山羊亚急性瘤胃酸中毒效果及作用机理[D].福州:福建农林大学, 2019. LI Y.The effects an mechanism of Hericium erinaceus polysaccharides mitigating SARA in goat[D].Fuzhou:Fujian Agriculture and Forestry University, 2019.(in Chinese)
[26] WANG L, LI Y, ZHANG Y, et al.The effects of different concentrate-to-forage ratio diets on rumen bacterial microbiota and the structures of Holstein cows during the feeding cycle[J].Animals, 2020, 10(6):957.
[27] SÖLLINGER A, TVEIT A T, POULSEN M, et al.Holistic assessment of rumen microbiome dynamics through quantitative metatranscriptomics reveals multifunctional redundancy during key steps of anaerobic feed degradation[J].mSystems, 2018, 3(4):10.
[28] 桂林生,陈渲,周力,等.日粮中添加酵母培养物对青海藏羊生长性能和瘤胃微生物多样性的影响[J].四川农业大学学报, 2023, 41(5):912-917. GUI L S, CHEN X, ZHOU L, et al.Effects of adding yeast cultures to diets on growth performance and rumen microbial diversity in Qinghai Tibetan sheep[J].Journal of Sichuan Agricultural University, 2023, 41(5):912-917.(in Chinese)
[29] REN Z, YAO R, LIU Q, et al.Effects of antibacterial peptides on rumen fermentation function and rumen microorganisms in goats[J].PLoS One, 2019, 14(8):e0221815.
[30] REN H, SU X, BAI H, et al.Specific enrichment of microbes and increased ruminal propionate production:The potential mechanism underlying the high energy efficiency of Holstein heifers fed steam-flaked corn[J].AMB Express, 2019, 9(1):209.