苜蓿干草与苜蓿青贮对奶牛瘤胃蛋白质利用的影响及作用机制的研究
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摘要
本研究通过5个试验系统探讨了日粮选用苜蓿干草或苜蓿青贮作为粗饲料,对奶牛瘤胃发酵、微生物蛋白合成、肽链降解酶浓度、瘤胃内微生物区系及蛋白质分解菌数量的影响,以期探究苜蓿不同加工形式产物(苜蓿干草vs.苜蓿青贮)对奶牛瘤胃内氮利用的影响及作用机制。
试验一选择苜蓿干草及精料作为试验饲料来源,以100%精料为对照组,将苜蓿干草以不同比例与精料混合(25：75、45：55、55：45和75：25),分析苜蓿干草与精粗比对体外瘤胃发酵、微生物蛋白含量、肽链内切酶和外切酶浓度及蛋白质降解菌数量的影响。将饲料样品粉碎后过1mm分析筛,作为短期人工瘤胃发酵底物,在恒温培养箱中(39℃)发酵24h,测定24h产气量、挥发性脂肪酸、氨态氮、微生物蛋白合成效率、肽链内切酶及外切酶浓度、总细菌及相关蛋白质降解细菌数量。结果表明：与全精料组相比,添加苜蓿干草,奶牛瘤胃乙酸／总挥发酸、氨态氮含量及总细菌数显著增加(P<0.05),24h产气量、总嘌呤含量、普雷沃氏菌及嗜淀粉瘤胃拟杆菌显著减少(P<0.05),肽链外切酶中的三肽基肽酶浓度显著降低(P<0.05)。精粗比对瘤胃内24h产气量及半胱氨酸蛋白酶浓度有显著降低的影响,随着苜蓿干草添加水平的增加,二者均呈线性下降(P<0.05)。苜蓿干草与精粗比二因素互作对本试验测定指标无显著影响,但对嗜淀粉瘤胃拟杆菌数量有极显著影响(P<0.01)。苜蓿干草：精料比例为55：45时,总细菌数、溶纤维丁酸弧菌及嗜淀粉瘤胃拟杆菌显著高于其他处理组(P<0.05)。
试验二选择苜蓿青贮及精料作为试验饲料来源,以100%精料为对照组,将苜蓿青贮以不同比例与精料混合(25：75、45：55、55：45和75：25),分析苜蓿青贮与精粗比对瘤胃发酵、微生物蛋白含量、肽链内切酶和外切酶浓度及蛋白质降解菌数量的影响。将饲料样品粉碎后过1mm分析筛,混匀后作为短期人工瘤胃发酵底物,在恒温培养箱中39℃条件下发酵24h,测定24h产气量、挥发性脂肪酸、氨态氮、微生物蛋白合成效率、肽链内切酶及外切酶、总细菌及与相关蛋白质降解菌。结果表明：与全精料组相比,添加苜蓿青贮,奶牛瘤胃24h产气量、微生物蛋白合成效率、天冬氨酸蛋白酶浓度、半胱氨酸蛋白酶浓度、二肽基肽酶浓度及普雷沃氏菌数均显著下降(P<0.05),氨态氮浓度、总细菌及溶纤维丁酸弧菌数显著增加(P<0.05)。随着苜蓿青贮比例增加,24h产气量、微生物蛋白合成效率、天冬氨酸蛋白酶浓度、半胱氨酸蛋白酶浓度、二肽基肽酶浓度均呈现显著降低的变化规律(P<0.05),氨态氮浓度线性增加(P<0.05)。氨态氮浓度、总细菌数、溶纤维丁酸弧菌及普雷沃氏菌数受到苜蓿青贮与精粗比互作的显著影响(P<0.05)。苜蓿青贮：精料比例为45：55时,溶纤维丁酸弧菌、牛链球菌、普雷沃氏菌及嗜淀粉瘤胃拟杆菌数最多。
试验三采集安徽省蚌埠地区苜蓿,分别加工为苜蓿干草及苜蓿青贮,将饲料样品粉碎过1mm分析筛,作为发酵底物在恒温培养箱中39℃条件下发酵24h,测定瘤胃液中挥发性脂肪酸、氨态氮、微生物蛋白合成效率、总细菌与相关蛋白质降解细菌数量。结果显示,苜蓿干草组总嘌呤含量(5.38mmol/L)显著高于(P<0.05)苜蓿青贮组(4.97mmol/L),两组之间氨态氮含量及微生物蛋白合成效率差异不显著,但苜蓿干草组氨态氮含量(26.51mg/l00mL)略低于苜蓿青贮组(27.59mg/l00mL),苜蓿干草组微生物蛋白合成效率(0.18)高于苜蓿青贮组(0.17),苜蓿干草组溶纤维丁酸弧菌数量显著高于苜蓿青贮组(P<0.05)。
试验四收集试验三中苜蓿干草与苜蓿青贮样品,粉碎过2.5mmm分析筛进行尼龙袋降解试验,比较二者的干物质、粗蛋白及18种氨基酸有效降解率。结果显示,苜蓿干草与苜蓿青贮之间干物质降解率差异不显著(P>0.05),苜蓿干草的粗蛋白有效降解率(41.18%)显著高于(P<0.05)苜蓿青贮的粗蛋白有效降解率(34.84%)。苜蓿青贮中的脯氨酸有效降解率(41.38%)略高于苜蓿干草中的脯氨酸降解率(P>0.05),但苜蓿干草中天冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、组氨酸、精氨酸、色氨酸及胱氨酸有效降解率均显著高于苜蓿青贮(P<0.05),苜蓿干草中赖氨酸及蛋氨酸有效降解率也高于苜蓿青贮(P>0.05)。
试验五收集苜蓿干草与苜蓿青贮经体外培养24h后的瘤胃液,采用高通量测序技术测定提取DNA后的微生物菌群分布,结果显示,苜蓿干草组中微生物OTUs数目高于苜蓿青贮组,苜两组之间微生物OTUs种类有较大的交叉度,聚类相似性阈值为90%时,苜蓿干草组含5386个OTUs,苜蓿青贮组含4633个OTUs,其中二者交叉3908个OTUs。聚类相似性阈值为97%时,苜蓿干草组含19544个OTUs,苜蓿青贮组含16099个OTUs,其中二者交叉8806个OTUS。瘤胃液中细菌种类较为相似,其中,拟杆菌属、梭状芽孢杆菌属丰度最高,球菌属、纤维弧菌及β-变形菌属丰度较高。
Five experiments were conducted to investigate the effects of alfalfa hay or haylage on ruminal fermentation, microbial synthesis, nitrogen utilization, difference of rumen bacteria flora in dairy cows, to explore possible mechanism on utilization of dietary protein in dairy cows with alfalfa hay and alfalfa silage.
Experiment1, Rumen fluids were collected from three Holstein heifers which fitted with permanent rumen fistulas, and an in vitro batch culture was conducted to investigate differences in the fermentation characteristics. Alfalfa hay (AH) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that acetate/total volatile acid ratio, ammonia nitrogen content and total bacterial count were significantly increased (P<0.05) with adding alfalfa hay; total purines content, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities significantly decreased (P<0.05) with alfalfa hay treatment, tripeptidyl-peptidase concentration also was affected by alfalfa hay (P<0.05). Cysteine peptidase concentration and24h gas production significantly increased with the increase of alfalfa hay level (P<0.05), the total bacteria, Butyrivibrio fibrisolvens, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities also affected by forage-to-concentrate ratio. Total bacterial count, Butyrivibrio fibrisolvens count and Ruminobacter amylophilus count were highest on55:45(AH: concentrate).
Experiment2, In vitro batch culture was conducted to investigate differences in the fermentation characteristics alfalfa silage (AS) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, Rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that24h gas production, efficiency of microbial protein synthesis, aspartic peptidase, cysteine peptidase, dipeptidyl-peptidase concentration and Prevotellabryantii gene expression quantity were significantly reduced (P<0.05) with alfalfa silage treatment; ammonia nitrogen concentration, total bacteria and Butyrivibrio fibrisolvens gene expression quantities significantly increased by alfalfa silage (P<0.05). Microbial protein synthesis, aspartic peptidase, cysteine peptidase concentration were significantly decrease, ammonia nitrogen concentration got higher (P<0.05) with the increase of alfalfa silage level (P<0.05). In this trial, ammonia nitrogen concentration, total bacteria, Butyrivibrio fibrisolvens and Prevotellabryantii gene expression quantities also affected by alfalfa silage and forage-to-concentrate ratio. Butyrivibrio fibrisolvens count, Streptococcus bovis count, Prevotellabryantii count and Ruminobacter amylophilus count were highest on45%alfalfa silage mixed55%concentrate.
Experiment3, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In vitro batch culture was conducted to investigate differences in the ammonia nitrogen, total purines and volatile fatty acid production and gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus between AH and AS. Rumen fluids were collected from three healthy Holstein heifers, fitted with permanent rumen fistulas. Results indicate that the total purines of AH was significantly higher (P><0.05) than those of AS, efficiency of microbial protein synthesis of AH also higher than that of AS even though there was not significantly affect (P>0.05).
Experiment4, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In situ ruminal dry matter, crude protein and amino acid degradation characteristics were determined for AH and AS with three Holstein heifers, fitted with permanent rumen fistulas. Effective crude protein degradability was significantly greater in AH than in AS (P<0.05). AH has higher aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isleucine, leucine, tyrosine, phenylalanine, histidine, arginine and tryptophan and cystine effective degradation rate (P<0.05) than AS. Lysine and methionine effective degradation rate in AH were higher than those in AS (P>0.05).
Experiment5, Collected rumen fluid with alfalfa hay or alfalfa silage were incubated24h by using in vitro batch culture, tested the microbial flora distribution by using high-throughput sequencing technology after DNA were extracted. Results show that microorganisms OTUs class of alfalfa hay group had large cross with that of alfalfa silage group. Microorganism OTUs number of AH was higher than that of AS, AHgroup contains5386OTUs, AS group contains4633OTUs, including both cross3908OTUs with the cutoff value of0.10. AH contains19544OTUs, AS contains16099OTUs, including both cross8806OTUs with the cutoff value of0.03. Thus, microbial flora class level in AH and that in AS was similar, bacteroides, clostridia, β-proteobacteria and fibrobacteria were major microorganisms.
试验一选择苜蓿干草及精料作为试验饲料来源,以100%精料为对照组,将苜蓿干草以不同比例与精料混合(25：75、45：55、55：45和75：25),分析苜蓿干草与精粗比对体外瘤胃发酵、微生物蛋白含量、肽链内切酶和外切酶浓度及蛋白质降解菌数量的影响。将饲料样品粉碎后过1mm分析筛,作为短期人工瘤胃发酵底物,在恒温培养箱中(39℃)发酵24h,测定24h产气量、挥发性脂肪酸、氨态氮、微生物蛋白合成效率、肽链内切酶及外切酶浓度、总细菌及相关蛋白质降解细菌数量。结果表明：与全精料组相比,添加苜蓿干草,奶牛瘤胃乙酸／总挥发酸、氨态氮含量及总细菌数显著增加(P<0.05),24h产气量、总嘌呤含量、普雷沃氏菌及嗜淀粉瘤胃拟杆菌显著减少(P<0.05),肽链外切酶中的三肽基肽酶浓度显著降低(P<0.05)。精粗比对瘤胃内24h产气量及半胱氨酸蛋白酶浓度有显著降低的影响,随着苜蓿干草添加水平的增加,二者均呈线性下降(P<0.05)。苜蓿干草与精粗比二因素互作对本试验测定指标无显著影响,但对嗜淀粉瘤胃拟杆菌数量有极显著影响(P<0.01)。苜蓿干草：精料比例为55：45时,总细菌数、溶纤维丁酸弧菌及嗜淀粉瘤胃拟杆菌显著高于其他处理组(P<0.05)。
试验二选择苜蓿青贮及精料作为试验饲料来源,以100%精料为对照组,将苜蓿青贮以不同比例与精料混合(25：75、45：55、55：45和75：25),分析苜蓿青贮与精粗比对瘤胃发酵、微生物蛋白含量、肽链内切酶和外切酶浓度及蛋白质降解菌数量的影响。将饲料样品粉碎后过1mm分析筛,混匀后作为短期人工瘤胃发酵底物,在恒温培养箱中39℃条件下发酵24h,测定24h产气量、挥发性脂肪酸、氨态氮、微生物蛋白合成效率、肽链内切酶及外切酶、总细菌及与相关蛋白质降解菌。结果表明：与全精料组相比,添加苜蓿青贮,奶牛瘤胃24h产气量、微生物蛋白合成效率、天冬氨酸蛋白酶浓度、半胱氨酸蛋白酶浓度、二肽基肽酶浓度及普雷沃氏菌数均显著下降(P<0.05),氨态氮浓度、总细菌及溶纤维丁酸弧菌数显著增加(P<0.05)。随着苜蓿青贮比例增加,24h产气量、微生物蛋白合成效率、天冬氨酸蛋白酶浓度、半胱氨酸蛋白酶浓度、二肽基肽酶浓度均呈现显著降低的变化规律(P<0.05),氨态氮浓度线性增加(P<0.05)。氨态氮浓度、总细菌数、溶纤维丁酸弧菌及普雷沃氏菌数受到苜蓿青贮与精粗比互作的显著影响(P<0.05)。苜蓿青贮：精料比例为45：55时,溶纤维丁酸弧菌、牛链球菌、普雷沃氏菌及嗜淀粉瘤胃拟杆菌数最多。
试验三采集安徽省蚌埠地区苜蓿,分别加工为苜蓿干草及苜蓿青贮,将饲料样品粉碎过1mm分析筛,作为发酵底物在恒温培养箱中39℃条件下发酵24h,测定瘤胃液中挥发性脂肪酸、氨态氮、微生物蛋白合成效率、总细菌与相关蛋白质降解细菌数量。结果显示,苜蓿干草组总嘌呤含量(5.38mmol/L)显著高于(P<0.05)苜蓿青贮组(4.97mmol/L),两组之间氨态氮含量及微生物蛋白合成效率差异不显著,但苜蓿干草组氨态氮含量(26.51mg/l00mL)略低于苜蓿青贮组(27.59mg/l00mL),苜蓿干草组微生物蛋白合成效率(0.18)高于苜蓿青贮组(0.17),苜蓿干草组溶纤维丁酸弧菌数量显著高于苜蓿青贮组(P<0.05)。
试验四收集试验三中苜蓿干草与苜蓿青贮样品,粉碎过2.5mmm分析筛进行尼龙袋降解试验,比较二者的干物质、粗蛋白及18种氨基酸有效降解率。结果显示,苜蓿干草与苜蓿青贮之间干物质降解率差异不显著(P>0.05),苜蓿干草的粗蛋白有效降解率(41.18%)显著高于(P<0.05)苜蓿青贮的粗蛋白有效降解率(34.84%)。苜蓿青贮中的脯氨酸有效降解率(41.38%)略高于苜蓿干草中的脯氨酸降解率(P>0.05),但苜蓿干草中天冬氨酸、苏氨酸、丝氨酸、谷氨酸、甘氨酸、丙氨酸、缬氨酸、异亮氨酸、亮氨酸、酪氨酸、苯丙氨酸、组氨酸、精氨酸、色氨酸及胱氨酸有效降解率均显著高于苜蓿青贮(P<0.05),苜蓿干草中赖氨酸及蛋氨酸有效降解率也高于苜蓿青贮(P>0.05)。
试验五收集苜蓿干草与苜蓿青贮经体外培养24h后的瘤胃液,采用高通量测序技术测定提取DNA后的微生物菌群分布,结果显示,苜蓿干草组中微生物OTUs数目高于苜蓿青贮组,苜两组之间微生物OTUs种类有较大的交叉度,聚类相似性阈值为90%时,苜蓿干草组含5386个OTUs,苜蓿青贮组含4633个OTUs,其中二者交叉3908个OTUs。聚类相似性阈值为97%时,苜蓿干草组含19544个OTUs,苜蓿青贮组含16099个OTUs,其中二者交叉8806个OTUS。瘤胃液中细菌种类较为相似,其中,拟杆菌属、梭状芽孢杆菌属丰度最高,球菌属、纤维弧菌及β-变形菌属丰度较高。
Five experiments were conducted to investigate the effects of alfalfa hay or haylage on ruminal fermentation, microbial synthesis, nitrogen utilization, difference of rumen bacteria flora in dairy cows, to explore possible mechanism on utilization of dietary protein in dairy cows with alfalfa hay and alfalfa silage.
Experiment1, Rumen fluids were collected from three Holstein heifers which fitted with permanent rumen fistulas, and an in vitro batch culture was conducted to investigate differences in the fermentation characteristics. Alfalfa hay (AH) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that acetate/total volatile acid ratio, ammonia nitrogen content and total bacterial count were significantly increased (P<0.05) with adding alfalfa hay; total purines content, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities significantly decreased (P<0.05) with alfalfa hay treatment, tripeptidyl-peptidase concentration also was affected by alfalfa hay (P<0.05). Cysteine peptidase concentration and24h gas production significantly increased with the increase of alfalfa hay level (P<0.05), the total bacteria, Butyrivibrio fibrisolvens, Prevotellabryantii and Ruminobacter amylophilus gene expression quantities also affected by forage-to-concentrate ratio. Total bacterial count, Butyrivibrio fibrisolvens count and Ruminobacter amylophilus count were highest on55:45(AH: concentrate).
Experiment2, In vitro batch culture was conducted to investigate differences in the fermentation characteristics alfalfa silage (AS) mixed concentrate with different ratio (0:100,25:75,45:55,55:45and75:25, respectively) in terms of24h gas production, volatile fatty acid production, ammonia nitrogen, total purines, efficiency of microbial protein synthesis, concentration of endopeptidase and exopeptidase, Rumen gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus. Results show that24h gas production, efficiency of microbial protein synthesis, aspartic peptidase, cysteine peptidase, dipeptidyl-peptidase concentration and Prevotellabryantii gene expression quantity were significantly reduced (P<0.05) with alfalfa silage treatment; ammonia nitrogen concentration, total bacteria and Butyrivibrio fibrisolvens gene expression quantities significantly increased by alfalfa silage (P<0.05). Microbial protein synthesis, aspartic peptidase, cysteine peptidase concentration were significantly decrease, ammonia nitrogen concentration got higher (P<0.05) with the increase of alfalfa silage level (P<0.05). In this trial, ammonia nitrogen concentration, total bacteria, Butyrivibrio fibrisolvens and Prevotellabryantii gene expression quantities also affected by alfalfa silage and forage-to-concentrate ratio. Butyrivibrio fibrisolvens count, Streptococcus bovis count, Prevotellabryantii count and Ruminobacter amylophilus count were highest on45%alfalfa silage mixed55%concentrate.
Experiment3, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In vitro batch culture was conducted to investigate differences in the ammonia nitrogen, total purines and volatile fatty acid production and gene expression quantities of total bacteria, Butyrivibrio fibrisolvens, Streptococcus bovis, Prevotellabryantii and Ruminobacter amylophilus between AH and AS. Rumen fluids were collected from three healthy Holstein heifers, fitted with permanent rumen fistulas. Results indicate that the total purines of AH was significantly higher (P><0.05) than those of AS, efficiency of microbial protein synthesis of AH also higher than that of AS even though there was not significantly affect (P>0.05).
Experiment4, Fresh alfalfa was harvested from AnHui province and then prepared as alfalfa hay (AH) and alfalfa silage (AS). In situ ruminal dry matter, crude protein and amino acid degradation characteristics were determined for AH and AS with three Holstein heifers, fitted with permanent rumen fistulas. Effective crude protein degradability was significantly greater in AH than in AS (P<0.05). AH has higher aspartic acid, threonine, serine, glutamic acid, glycine, alanine, valine, isleucine, leucine, tyrosine, phenylalanine, histidine, arginine and tryptophan and cystine effective degradation rate (P<0.05) than AS. Lysine and methionine effective degradation rate in AH were higher than those in AS (P>0.05).
Experiment5, Collected rumen fluid with alfalfa hay or alfalfa silage were incubated24h by using in vitro batch culture, tested the microbial flora distribution by using high-throughput sequencing technology after DNA were extracted. Results show that microorganisms OTUs class of alfalfa hay group had large cross with that of alfalfa silage group. Microorganism OTUs number of AH was higher than that of AS, AHgroup contains5386OTUs, AS group contains4633OTUs, including both cross3908OTUs with the cutoff value of0.10. AH contains19544OTUs, AS contains16099OTUs, including both cross8806OTUs with the cutoff value of0.03. Thus, microbial flora class level in AH and that in AS was similar, bacteroides, clostridia, β-proteobacteria and fibrobacteria were major microorganisms.
引文
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