日粮中添加小肽对育肥牦牛生产性能和消化道PepT1 mRNA表达的影响

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苗建军^,, 彭忠利^,, 高彦华, 柏雪, 谢昕廷西南民族大学畜牧兽医学院/青藏高原动物遗传资源保护与利用教育部重点实验室,成都 610041

Effects of Dietary Small Peptides on Production Performance and Expression of PepT1 mRNA in Digestive Tract of Fattening Yaks

MIAO JianJun^,, PENG ZhongLi^,, GAO YanHua, BAI Xue, XIE XinTingCollege of Animal and Veterinary Sciences, Southwest Minzu University/Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization, Ministry of Education, Chengdu 610041

通讯作者: 彭忠利,E-mail： leo3131@163.com

责任编辑: 林鉴非
收稿日期:2019-12-11接受日期:2020-05-12网络出版日期:2020-12-01

基金资助:

国家重点研发计划.2017YFC0504806
国家现代农业产业技术体系四川省肉牛创新团队.sccxtd-2020-13
四川省重点研发项目.2017NZ0042

Received:2019-12-11Accepted:2020-05-12Online:2020-12-01
作者简介 About authors
苗建军,E-mail： 332779037@qq.com。

摘要
【目的】研究日粮中添加小肽对育肥牦牛生产性能、养分表观消化率、血液指标及消化道小肽转运载体1（PepT1）mRNA表达的影响,为小肽在牦牛日粮中的合理应用提供数据支持。【方法】选取36头体重（（180.98±20.57）kg）相近,健康的麦洼公牦牛,按照随机区组试验设计分为4组,每组9个重复,每个重复1头牛。分别饲喂小肽添加水平为0、0.75%、1.50%、2.25%的全混合日粮。预饲期30 d,正试期80 d。试验开始和结束时记录体重,每日饲喂时记录每头牦牛的喂料量及剩料量。试验最后一周,连续3 d收集每头牦牛的饲料及粪便样品,进行常规营养分析;同时,每组选取5头牦牛采集颈静脉血并制备血清,测定血清生化及免疫指标。试验结束后,将每组采血的5头牦牛屠宰,立即取瘤胃、网胃、瓣胃、皱胃及十二指肠、空肠、回肠组织,通过实时荧光定量PCR法测定各组织中PepT1 mRNA的表达量。【结果】（1）平均日增重（ADG）、干物质采食量（DMI）和料重比（F/G）随小肽添加水平的升高均呈二次曲线变化（P<0.05）,以2.25%组牦牛的生产性能表现最优,其DMI和ADG均显著高于对照组和0.75%组（P<0.05）,而F/G显著低于对照组和0.75%组（P<0.05）。（2）随小肽添加水平的升高,日粮有机物（OM）和粗蛋白（CP）的表观消化率呈二次曲线上升（P<0.05）,中性洗涤纤维（NDF）和酸性洗涤纤维（ADF）的表观消化率线性升高（P<0.01）,以2.25%组消化率最高;各处理组中钙、磷的表观消化率无显著差异（P>0.05）。（3）随小肽添加水平的升高,血清中谷丙转氨酶（ALT）含量线性降低（P<0.05）,而尿素氮（UN）含量线性升高（P<0.05）,总蛋白（TP）含量有线性升高的趋势（P<0.10）,谷草转氨酶（AST）含量呈二次曲线降低（P<0.05）,而碱性磷酸酶（ALP）、葡萄糖（GLU）、胆固醇（CHO）、甘油三酯（TG）含量未产生显著变化（P>0.05）;日粮中添加不同水平小肽对牦牛血清中IgG、IgA和IgM三种免疫球蛋白的含量均无显著影响（P>0.05）。（4）牦牛消化道中PepT1 mRNA表达量由高到低依次为空肠、回肠、十二指肠、网胃、瓣胃、瘤胃、皱胃,且空肠PepT1 mRNA的表达量显著高于其他消化道部位（P<0.05）,而皱胃PepT1 mRNA的表达量显著低于空肠和回肠（P<0.05）;回肠、十二指肠、瘤胃、网胃、瓣胃的PepT1 mRNA表达量无显著差异（P>0.05）。随小肽添加水平的升高,瘤胃、网胃和空肠的PepT1 mRNA表达量均呈线性升高变化（P<0.05）,但对瓣胃、皱胃、十二指肠和回肠的PepT1 mRNA表达量均无显著影响（P>0.05）。【结论】日粮中添加小肽能够提高育肥牦牛的生产性能和养分表观消化率,改善肝功能,促进小肽在消化道中的转运吸收。在本试验条件下,日粮小肽添加水平为2.25%时饲喂效果最佳。
关键词： 小肽;育肥牦牛;生产性能;养分表观消化率;血液指标;PepT1 mRNA表达

Abstract

【Objective】This study investigated the effects of dietary small peptides on production performance, apparent digestibility of nutrients, blood indexes and expression of PepT1 mRNA in digestive tract of fattening yaks. 【Method】Health Maiwa male yaks (n=36, (180.98±20.57) kg body weight) were divided into 4 groups in a randomized complete block design, and each group had 9 male yaks. And they were fed 4 types of total mixed ratio diet with small peptides additions of 0, 0.75%, 1.50% and 2.25%, respectively. There was a pre-experimental period of 30 days, followed by a trial period of 80 days. Every yak’s weight was recorded before and after the trial begins, and the feed intake was also recorded every day. During the last week of the trial period, the fecal grab samples were collected for 3 consecutive days from each yak for routine analysis of nutrients. And five yaks were selected from each group to collect jugular vein blood and prepare serum to determine serum biochemical and immune indicators. At the end of the trial period, 5 yaks with blood collected were slaughtered, and the rumen, reticulum, omasum, abomasum, duodenum, jejunum, and ileum tissue were collected to determine the expression of PepT1 mRNA by using real time quantitative PCR. 【Result】(1) With the level of small peptides increasing, the dry matter intake, average daily gain and the feed gain ratio showed quadratic variation (P<0.05). The group with the best production performance was the 2.25% group. Compared with the control group and the 0.75% group, the dry matter intake and average daily gain were significant improved in the 2.25% group, but the feed gain ratio was significant decreased in the 2.25% group. (2) With increasing of small peptides levels, the degradation rate of neutral detergent fibre and acid detergent fibre linearly increased (P<0.01); the degradation rate of organic matter and crude protein presented quadratic variation (P<0.05). The 2.25% group had the highest digestibility. No significant difference existed between the degradation rate of calcium and phosphorus in four groups (P>0.05). (3) Across the range of small peptides supplementation levels, the content of alanine aminotransferase linearly decreased (P<0.05); the content of urea nitrogen linearly increased (P<0.05); the total protein showed linearly increased trend (P<0.10), and the content of aspartate aminotransferase showed quadratic variation (P<0.05). There were no significant effects on the content of alkaline phosphatase, glucose, cholesterol, triglyceride and immunoglobulins (IgG, IgA and IgM) in yak serum by adding different levels of small peptides. (4) The expression of PepT1 mRNA in the digestive tract of yaks from the highest to the lowest was the jejunum, ileum, duodenum, reticulum, omasum, rumen, and abomasum, while the expression of PepT1 mRNA in jejunum was significantly higher than that of the other gastrointestinal tracts (P<0.05). The expression of PepT1 mRNA in abdomen was significantly lower than those of jejunum and ileum. No significant difference was found between the expression of PepT1 mRNA in ileum, duodenum, reticulum, omasum, and rumen. With increasing of small peptides levels from 0 to 2.25%, the expression of PepT1 mRNA in rumen, reticulum and jejunum linearly increased (P<0.05), and small peptides levels had no significant effect on the expression of PepT1 mRNA in omasum, abomasum, duodenum and ileum (P>0.05). 【Conclusion】 In conclusion, dietary small peptides could improve the production performance, apparent digestibility of nutrients, liver function of fattening yaks, and promote the transport and absorption of small peptides in the digestive tract. In this research, the appropriate level of dietary small peptides of yaks was 2.25%.

Keywords：small peptides;fattening yaks;production performance;apparent digestibility of nutrients;blood indexes;expression of PepT1 mRNA

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本文引用格式
苗建军, 彭忠利, 高彦华, 柏雪, 谢昕廷. 日粮中添加小肽对育肥牦牛生产性能和消化道PepT1 mRNA表达的影响[J]. 中国农业科学, 2020, 53(23): 4950-4960 doi:10.3864/j.issn.0578-1752.2020.23.019
MIAO JianJun, PENG ZhongLi, GAO YanHua, BAI Xue, XIE XinTing. Effects of Dietary Small Peptides on Production Performance and Expression of PepT1 mRNA in Digestive Tract of Fattening Yaks[J]. Scientia Agricultura Sinica, 2020, 53(23): 4950-4960 doi:10.3864/j.issn.0578-1752.2020.23.019

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0 引言

【研究意义】受自然条件、风俗习惯等影响,青藏高原地区的牦牛养殖长久以来处于靠天养畜的传统模式下,集约化程度低,生产效率低下。牦牛舍饲育肥技术的推广,能够提高牦牛的生产效率,弥补牦牛肉在市场上断季供应的缺陷,提升牦牛产品的商品率。因此,选择适宜饲料添加剂,优化牦牛饲料配方,对于提升牦牛的生产性能,增加牧民的经济效益具有重要意义。【前人研究进展】小肽是含有2或3个氨基酸的小分子物质,主要由饲粮蛋白在胃肠道内消化后得到,具有多种生物学功能,是一种安全、高效的饲料添加剂^[1]。有研究表明,在日粮中添加小肽能够促进动物机体蛋白质的合成^[2]、增强机体免疫力^[3,4,5]、提高饲料养分的消化率^[6]、提高动物的生产性能^[7,8,9],具有广阔的应用前景。小肽的转运方式主要包括：载体转运、渗透扩散、旁细胞穿透和胞吞4种方式,载体转运为主动转运,需要消耗能量,其余3种方式为被动转运,无能量消耗^[10]。转运载体主要为小肽转运载体1（PepT1）,可以转运二肽、三肽和拟肽类药物^[1]。区别于氨基酸的转运载体,PepT1的转运不存在专一性,对400种二肽和8 000种三肽具有转运能力,是低亲和力、高容量的载体^[11]。反刍动物PepT1主要分布于胃上皮组织和小肠黏膜^[12,13],在奶牛^[14]、绵羊^{[12, 15]}、湖羊^[16]上的研究显示,PepT1 mRNA在小肠各肠段的表达量要高于胃,但在肉牛上的研究发现,PepT1 mRNA在瓣胃和瘤胃的表达量要高于十二指肠^[13],可见PepT1 mRNA在消化道中的表达存在物种差异性。【本研究切入点】牦牛是高海拔地区的特有家畜,其对氮利用的机制可能与其他低海拔生存的反刍动物有所不同,且饲养方式的改变能否引起PepT1表达的差异也有待进一步研究。【拟解决的关键问题】本研究通过开展牦牛的舍饲育肥试验,探讨日粮中添加不同水平小肽对育肥牦牛生产性能、养分表观消化率以及血清生化指标的影响,并通过实时荧光定量PCR技术,对牦牛消化道PepT1 mRNA的表达量进行分析,旨在揭示PepT1在育肥牦牛消化道的表达特性,并探寻小肽添加水平对牦牛消化道PepT1表达的影响,为小肽在牦牛日粮中的合理应用提供数据支持。

1 材料与方法

1.1 试验材料

材料：富力肽[酸溶蛋白（小肽）≥28.0%,水分≥8.0%,粗脂肪≥2.5%,粗灰分≥15.0%,粗纤维≥7.0%,游离棉酚≤400 mg·kg^-1],由成都美溢德生物技术有限公司生产,选用棉籽蛋白为原料,经液态组合酶解制得（表1）。

Table 1
表1
表1富力肽中氨基酸组成及含量（干物质基础）
Table 1Composition and content of amino acid in the peptide (DM basis, %)

氨基酸 Amino acid	含量Content（%）	氨基酸 Amino acid	含量 Content（%）
缬氨酸Valine	2.27	赖氨酸Lysine	2.52
亮氨酸Leucine	2.76	组氨酸Histidine	1.28
异亮氨酸Isoleucine	1.60	苏氨酸Threonine	1.40
蛋氨酸Methionine	0.66	甘氨酸Glicine	2.27
胱氨酸Cystine	0.85	丙氨酸Alanine	1.86
苯丙氨酸Phenylalanine	2.25	脯氨酸Proline	1.94
酪氨酸Tyrosine	1.21	丝胺酸Serine	1.75
色氨酸Tryptophan	0.44	谷氨酸Glutamic	4.20
精氨酸Arginine	4.74	天冬酰胺Asparagine Acid	9.55

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1.2 试验时间与地点

试验于2017年11月至2018年3月在四川省阿坝藏族羌族自治州茂县茂欣农牧业发展有限责任公司进行,试验场海拔1 450 m,整个试验期内圈舍最高温25℃,最低温4℃。

1.3 试验动物与试验设计

选取36头体重（（180.98±20.57）kg）相近,健康的麦洼公牦牛,按照随机区组试验设计分为4组,每组9个重复,每个重复1头牛。分别饲喂小肽添加水平为0、0.75%、1.50%、2.25%的全混合日粮。预饲期30 d,正试期80 d。

1.4 试验日粮

采用TMR全混合日粮,由玉米型精料和青贮、发酵酒糟混合而成。每天按预估采食量现配现用。各处理组日粮组成及营养成分见表2。

Table 2
表2
表2试验日粮组成及营养成分（干物质基础）
Table 2The diet composition and nutrient components (DM basis, %)

原料 Ingredient	小肽添加水平 Addition levels of small peptide
原料 Ingredient	0	0.75%	1.50%	2.25%
玉米Corn	35.00	35.00	35.00	35.00
豆粕Soybean meal	7.00	7.00	7.00	7.00
玉米酒精糟及可溶物 Distiller dried grains with solubles	4.70	3.95	3.20	2.45
磷脂粉Phosphatide powder	0.75	0.75	0.75	0.75
碳酸钙 CaCO₃	0.60	0.60	0.60	0.60
氯化钠NaCl	0.50	0.50	0.50	0.50
碳酸氢钠 NaHCO₃	0.50	0.50	0.50	0.50
预混料¹⁾ Premix	0.95	0.95	0.95	0.95
小肽 Small peptide	0	0.75	1.50	2.25
发酵酒糟 Fermented distillers^,grains	25.00	25.00	25.00	25.00
玉米秸秆青贮Corn straw silage	25.00	25.00	25.00	25.00
合计Total	100.00	100.00	100.00	100.00
营养水平²⁾ Nutrient levels
干物质 DM	69.62	69.63	69.65	69.64
增重净能 Neg (MJ·kg^--1)	4.63	4.63	4.64	4.64
粗蛋白 CP	14.54	14.56	14.60	14.63
粗脂肪 EE	3.05	3.04	3.05	3.06
中性洗涤纤维 NDF	30.04	29.94	29.87	29.76
酸性洗涤纤维 ADF	16.68	16.59	16.48	16.38
钙 Ca	0.65	0.64	0.64	0.64
总磷 P	0.52	0.51	0.51	0.51

¹⁾预混料为每千克日粮提供：维生素A 2 500 IU,维生素D 500 IU,维生素E 30 IU,铜10 mg,铁50 mg,锰30 mg,锌30mg,碘0.5 mg,硒0.1 mg,钴0.2 mg。²⁾ 增重净能为计算值,其他营养指标为实测值
¹⁾The premix provided the following per kg of diets：VA 2500 IU, VD 500 IU, VE 30 IU, Cu 10 mg, Fe 50 mg, Mn 30 mg, Zn 30 mg, I 0.5 mg, Se 0.1 mg, Co 0.2 mg. ²⁾ Net energy for gain was a calculated value, while other nutrient indexes were measured values

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1.5 饲养管理

试验开始前对圈舍清理消毒,牦牛进圈后用伊维菌素注射液皮下注射驱虫,称重后分组,栓系饲喂,每日8：00和15：00各饲喂一次,自由采食、自由饮水。

1.6 样品采集及检测指标

1.6.1 生产性能试验开始和结束时,晨饲前对每头试验牦牛空腹称重,记录初重（IBW）和末重（FBW）;每日饲喂时,精确记录每头牦牛的喂料量及剩料量,计算干物质采食量,试验结束后计算平均日增重（ADG）、平均干物质采食量（DMI）以及料重比（F/G）。

1.6.2 养分表观消化率试验结束前3天,每天每头牦牛收集新鲜粪便100 g,每份粪便加入10 mL 10%的H₂SO₄固氮,3 d收集完成后,将同一头牦牛的粪便混合,取150 g于-20℃冷冻保存。除粪样外,每天同时采集各处理组日粮1 kg,将3 d的样品混合后取样1 kg,带回实验室65℃烘干,粉碎后过20目筛,进行常规营养分析,采用酸不溶灰分（AIA）作为指示剂,计算各养分的表观消化率。

样品中的干物质（DM）、粗蛋白（CP）、钙（Ca）、磷（P）的测定参照张丽英^[17]的方法,中性洗涤纤维（NDF）和酸性洗涤纤维（ADF）的测定参照VAN SOEST等^[18]的方法,有机物（OM）通过计算得出,酸不溶灰分的测定参照GB/T 23742-2009的方法。各养分表观消化率计算公式如下：

某养分表观消化率= 100%-（饲料中AIA含量/粪中AIA含量）×（粪中某养分含量/饲料中某养分含量）×100%

1.6.3 血清生化及免疫指标试验第80天,每组随机选取5头牦牛,对每头牦牛空腹颈静脉采血20 mL,然后在4℃下4 000 r/min离心5 min收集血清样品,分装于3个1.5 mL EP管后,-20℃保存。用全自动生化分析仪（TC6010L,江西）测定血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（ALP）、尿素氮（UN）、总蛋白（TP）、葡萄糖（GLU）、甘油三酯（TG）、胆固醇（CHO）含量。采用南京建成生物工程研究所试剂盒测定血清免疫球蛋白A（IgA）、免疫球蛋白G（IgG）和免疫球蛋白M（Ig M）含量。

1.6.4 消化道PepT1 mRNA表达量的测定试验结束后,所有试验牦牛分批运送至屠宰场待宰。将每组采血的5头牦牛,经电击、放血、剥皮后,将内脏分离,立即取瘤胃、网胃、瓣胃、皱胃、十二指肠、空肠、回肠组织,用冰生理盐水清洗内容物。在洁净的托盘上剥离瘤胃、网胃、瓣胃上皮组织,并刮取皱胃黏膜和十二指肠、空肠、回肠的肠道黏膜,各部位取2块,并用锡箔纸包好,放入2 mL的冻存管中,立即投入液氮带回实验室保存。

依照Rayscript cDNA Synthesis KIT试剂盒使用说明书,将提取的各上皮组织和黏膜的总RNA反转录为cDNA。以Pep T1为目的基因,由上海捷瑞生物公司合成引物（表3）,经荧光定量PCR方法对牦牛消化道样本进行PepT1 mRNA定量分析;反应体系为：SYBR Green Mix 7 μL,上下游引物各0.5 μL,cDNA 8 μL;条件为95℃预变性5 min,95℃变性10 s,60℃退火34 s,共40个循环。

Table 3
表3
表3引物序列
Table 3Primer sequence

目的基因 Target gene	引物名称 Primer name	引物序列 Primer sequence	片段大小 Fragment size（bp）
Pep T1	PepT1_2F	TGTCGCTGTCCATCGTCTA	78
	PepT1_2R	GGTTGAAGTCCGTGAGGTC
18S	18S_1F	CAACACGGGAAACCTCACC	118
	18S_1R	CCCAGACAAATCGCTCCAC

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1.7 数据分析

各样品PepT1 mRNA表达量的计算采用2^-??Ct法,以0水平组中瘤胃PepT1 mRNA表达量作为对照。数据通过Excel 2010处理后,再使用SPSS 24.0中的GLM模型进行单因素方差分析,采用Duncan’s法多重比较,随后使用曲线估计（Curve estimation）进行线性（Linear）和二次函数（Quadratic）的显著性检验。结果以平均值±标准差的形式表示,P<0.01表示差异极显著,P<0.05表示差异显著,P>0.05表示差异不显著。

2 结果

2.1 小肽添加水平对育肥牦牛生产性能的影响

由表4可知,随小肽添加水平的升高,ADG、DMI和F/G呈二次曲线变化（P<0.05）,2.25%组的DMI和ADG显著高于对照组和0.75%组（P<0.05）,而F/G显著低于对照组和0.75%组（P<0.05）;FBW有线性增高的趋势（P<0.10）,以2.25%组牦牛的生产性能表现最优。

Table 4
表4
表4小肽添加水平对育肥牦牛生产性能的影响
Table 4Effects of dietary small peptides levels on production performance of fattening yaks

项目 Items	小肽添加水平Addition levels of small peptide				P值 P value
项目 Items	0	0.75%	1.50%	2.25%	T	L	Q
初重IBW (kg)	180.67±27.98	180.22±28.51	181.25±33.38	181.78±27.97	1.000	0.919	0.994
末重FBW (kg)	230.00±34.54	227.56±35.25	238.75±26.74	257.11±31.77	0.224	0.060	0.110
平均日增重ADG (kg·d^-1)	0.64±0.15B	0.61±0.13B	0.72±0.13B	0.91±0.09A	0.001	<0.001	<0.001
干物质采食量DMI (kg·d^-1)	4.84±0.59b	4.79±0.41b	5.12±0.44ab	5.48±0.56a	0.024	0.005	0.009
料重比F/G	8.07±1.72a	8.32±1.86a	7.35±1.59ab	5.95±0.72b	0.014	0.004	0.005

同行数据不同大写字母表示差异极显著（P<0.01）,不同小写字母表示差异显著（P<0.05）,相同或者无字母表示差异不显著（P>0.05）,下同。T表示不同处理,L表示一次线性,Q表示二次曲线。下同
In the same row, values with no letters or the same letters mean no significant at P>0.05, while with different small letters mean significant difference at P<0.05, with different capital letters mean significant difference at P<0.01, the same below. T means Treatment, L means Linear, Q means Quadratic. The same below

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2.2 小肽添加水平对育肥牦牛养分表观消化率的影响

由表5可知,随小肽添加水平的升高,OM和CP的表观消化率呈二次曲线变化（P<0.05）,1.50%组和2.25%组对OM的消化率显著高于0.75%组（P<0.05）,2.25%组中CP的消化率显著高于对照组和0.75%组（P<0.05）;NDF和ADF的表观消化率线性升高（P<0.01）,1.50%组和2.25%组NDF和ADF的消化率显著高于对照组和0.75%组（P<0.01）,以2.25%组养分消化率最优。不同处理组间钙、磷的表观消化率均无显著差异（P>0.05）。

Table 5
表5
表5小肽添加水平对育肥牦牛养分表观消化率的影响
Table 5Effects of dietary small peptides levels on nutrients apparent digestibility of fattening yaks (%)

项目 Items	小肽添加水平Addition levels of small peptide				P值 P value
项目 Items	0	0.75%	1.50%	2.25%	T	L	Q
有机物OM	64.91±0.86ab	63.34±2.25b	65.71±1.03a	67.13±1.55a	0.011	0.016	0.010
蛋白质CP	51.30±3.61b	50.71±3.81b	53.29±1.42ab	54.11±2.25a	0.025	0.006	0.019
酸性洗涤纤维ADF	47.11±2.60B	49.19±3.40B	53.04±3.83A	54.02±1.86A	0.002	<0.001	<0.001
中性洗涤纤维NDF	50.34±1.12C	51.55±1.52C	55.03±2.83B	58.62±0.76A	0.001	<0.001	<0.001
钙Ca	58.08±4.53	57.01±4.39	60.21±4.60	59.50±3.63	0.374	0.373	0.680
磷P	53.61±5.75	53.61±0.82	54.39±5.10	54.07±8.51	0.996	0.853	0.981

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2.3 小肽添加水平对育肥牦牛血清生化及免疫指标的影响

由表6可知,随小肽添加水平的升高,血清中ALT含量线性降低（P<0.05）,而UN含量线性升高（P<0.05）,TP含量有线性升高的趋势（P<0.10）,AST含量呈二次曲线变化（P<0.05）,且1.50%组和2.25%组AST含量显著低于对照组和0.75%组（P<0.05）。日粮中添加不同水平小肽对血清中ALP、GLU、CHO和TG含量均无显著影响（P>0.05）。

Table 6
表6
表6小肽添加水平对育肥牦牛血清生化指标的影响
Table 6Effects of dietary small peptides levels on serum metabolites of fattening yaks

项目 Items	小肽添加水平Addition levels of small peptide				P值 P value
项目 Items	0	0.75%	1.50%	2.25%	T	L	Q
谷丙转氨酶ALT (U/L)	39.88±5.54	35.60±3.14	33.95±1.45	32.37±3.87	0.099	0.013	0.039
谷草转氨酶AST (U/L)	80.45±13.02a	79.50±12.66a	59.00±5.64b	63.05±8.35b	0.024	0.011	0.040
碱性磷酸酶ALP (U/L)	157.23±28.74	138.25±15.03	134.80±21.98	135.48±15.87	0.480	0.192	0.284
总蛋白TP (g·L^-1)	70.60±6.95	73.43±1.82	76.10±2.79	75.98±2.87	0.322	0.068	0.165
尿素氮UN (mmol·L^-1)	5.15±0.86	5.53±0.61	5.70±0.68	6.44±0.85	0.148	0.022	0.070
葡萄糖GLU (mmol·L^-1)	4.63±0.21	4.63±0.18	4.66±0.36	4.86±0.39	0.686	0.294	0.471
胆固醇CHO (mmol·L^-1)	2.14±0.37	2.05±0.13	1.92±0.35	1.88±0.14	0.790	0.298	0.587
甘油三酯TG (mmol·L^-1)	0.33±0.05	0.33±0.10	0.32±0.03	0.29±0.07	0.763	0.312	0.548

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由表7可知,日粮中添加不同水平小肽对育肥牦牛血清中IgG、IgA和IgM 3种免疫球蛋白的含量均无显著影响（P>0.05）。

Table 7
表7
表7小肽添加水平对育肥牦牛血清免疫指标的影响
Table 7Effects of dietary small peptides levels on serum immunity indexes of fattening yaks

项目 Items (mg·mL^-1)	小肽添加水平Addition levels of small peptide				P值 P value
项目 Items (mg·mL^-1)	0	0.75%	1.50%	2.25%	T	L	Q
IgG	13.78±1.37	14.01±1.79	14.40±1.20	14.39±1.49	0.899	0.481	0.744
IgA	6.95±2.57	6.64±1.73	6.10±0.64	7.24±1.54	0.871	0.810	0.723
IgM	1.56±0.19	1.49±0.18	1.79±0.09	1.59±0.18	0.196	0.539	0.506

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2.4 育肥牦牛消化道PepT1 mRNA的组织表达特性

由图1可知,牦牛消化道中PepT1 mRNA表达量由高到低依次为空肠、回肠、十二指肠、网胃、瓣胃、瘤胃、皱胃,且空肠的Pep T1 mRNA表达量显著高于其他消化道部位（P<0.05）,而皱胃PepT1 mRNA的表达量显著低于空肠和回肠（P<0.05）。回肠、十二指肠、瘤胃、网胃、瓣胃的PepT1 mRNA表达量无显著差异（P>0.05）。

图1

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图1育肥牦牛消化道Pep T1 mRNA的组织表达特性

柱形标注不同字母表示差异显著（P<0.05）
Fig. 1Tissue expression characteristics of PepT1 mRNA in the digestive tract of fattening yaks

Columns with different letter mean significant difference（P<0.05）

2.5 小肽添加水平对育肥牦牛消化道PepT1 mRNA表达量的影响

由表8可知,随小肽添加水平的升高,瘤胃、网胃和空肠的PepT1 mRNA表达量均呈线性升高变化（P<0.05）,但小肽添加水平对瓣胃、皱胃、十二指肠和回肠的PepT1 mRNA表达量无显著影响（P>0.05）。

Table 8
表8
表8小肽添加水平对育肥牦牛消化道PepT1 mRNA表达量的影响
Table 8Effects of dietary small peptides levels on the expression of PepT1 mRNA in the digestive tract of fattening yaks

项目 Items	小肽添加水平Addition levels of small peptide				P值 P value
项目 Items	0	0.75%	1.50%	2.25%	T	L	Q
瘤胃Rumen	0.79±0.31b	0.86±0.19b	1.31±0.92b	2.44±1.07a	0.028	0.006	0.009
网胃Reticulum	0.82±0.39	1.06±0.52	1.82±0.96	2.31±1.20	0.101	0.011	0.043
瓣胃Omasum	1.44±0.80	1.37±0.34	1.42±0.46	1.28±0.36	0.979	0.739	0.944
皱胃Abomasum	1.15±0.69	0.72±0.25	0.83±0.39	0.75±0.29	0.882	0.552	0.768
十二指肠Duodenum	2.19±0.32	3.31±0.97	2.14±0.59	2.08±0.56	0.318	0.546	0.469
空肠Jejunum	8.61±2.92	9.17±3.80	14.73±4.95	16.74±5.09	0.109	0.016	0.059
回肠Ileum	3.98±0.71	4.57±0.91	4.97±0.89	4.39±0.37	0.739	0.602	0.537

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3 讨论

3.1 小肽添加水平对育肥牦牛生产性能的影响

日粮中加入小肽能够提高动物的生产性能。祝平^[7]的研究发现,在日粮中添加2%、4%的植物小肽能够降低断奶仔猪的料重比和腹泻指数,提高日增重,但对采食量无显著影响。卜艳玲等^[9]报道称,在断奶仔猪日粮中添加肠杆菌肽能显著提高日增重,降低料重比。宋增廷^[6]在育肥羊的日粮中分别添加500和800 mg·kg^-1的谷胱甘肽,结果显示,与对照组相较,两试验组均显著提高了日增重,降低了料重比。本研究中,当小肽添加水平达到2.25%时,牦牛的ADG和DMI比对照组显著提高,料重比显著降低,生长性能最佳。1.50%和2.25%组采食量的升高,说明小肽能够改善日粮的适口性,提升牦牛的采食量,提高能量和蛋白的摄入量,增加体重。另有研究表明,小肽能够提高肌肉中胰岛素样生长因子-1（IGF-1）mRNA的表达量,促进生长轴中生长激素（GH）与IGF-1的分泌^[6],本试验中牦牛体重的显著增加也可能与此有关。但前期的体外试验表明,当小肽的添加量超过2.25%时会产生负效应,而在实际的生产当中是否存在同样的效果,还有待进一步研究。

3.2 对育肥牦牛养分表观消化率的影响

瘤胃中发酵非结构性碳水化合物的微生物能够利用肽作为氮源^[19],外源小肽的加入,能够刺激该类瘤胃微生物的生长,加速对淀粉、糖类等的发酵,促进瘤胃对营养成分的降解。另有研究表明,小肽能够提高瘤胃中纤维分解菌的活力,促进对纤维素和半纤维素的降解^[20,21]。王文娟^[22]分别给安装永久性瘘管的鲁西黄牛每天灌注100、200、300 g大豆小肽,结果显示,小肽的灌注,提高了机体对DM、OM、NDF、ADF和氮的表观消化率。在本研究中,日粮中加入小肽,促进了牦牛瘤胃对OM、CP、NDF、ADF的消化率,与上述报道结果相似。本课题组对瘤胃菌群的研究结果显示,小肽能够提高普雷沃氏菌属、丁酸弧菌属和拟杆菌属的相对丰度,而普雷沃氏菌属和丁酸弧菌属对蛋白、淀粉等均有降解作用,拟杆菌属能够促进纤维类物质的消化,几种菌属丰度的提高能够促进饲粮成分在瘤胃中的消化利用。

3.3 对育肥牦牛血清生化及免疫指标的影响

血清指标能够反映机体的代谢和病理状况。TP含量能够衡量机体蛋白质的吸收和代谢状况,TP含量的升高,可以促进组织蛋白的沉积。本试验中,随小肽水平的升高,TP含量有线性升高的趋势,有利于机体组织蛋白的沉积。血清中ALT和AST能够反映肝脏受损状况,当肝脏受损时,两种转氨酶的活性会迅速增高。在本试验中发现,ALT含量随小肽水平升高线性降低,1.50%和2.25%组牦牛血清中AST的活性显著降低,与孙海元的报道相近,小肽能够使肝脏功能得到改善,减轻肝脏损伤^[23]。CHO能够储存能量,但过高的CHO会引发心血管等疾病,本研究中CHO和TG含量在各组之间无显著差异,表明小肽没有带来负面影响。GLU能够反映机体对能量的调节是否恒定,有研究表明,动物血清中GLU的含量不应超过6.1 mmol·L^{-1 [24]},本研究中,各组GLU含量均在4.6—4.9 mmol·L^-1之间变动,未超过上述界限,且各组之间无显著差异,供能均衡。UN能够反映动物机体蛋白质代谢和氨基酸之间的平衡状况。UN含量过高,说明氨基酸平衡受到影响,氮代谢效率降低,本研究中,随小肽水平的升高,UN含量线性升高,与SWANSON等^[25]在羔羊上的研究相近,但本研究中各组之间UN含量无显著差异,未对机体氨基酸平衡造成显著不利影响。

IgA、IgG、IgM 3种免疫球蛋白参与机体的体液免疫,其含量能够反映机体免疫力的强弱。张迪等^[26]的研究表明,在种公猪日粮中分别添加1%、2%的小肽对血清中IgG、IgA、IgM的含量无显著影响;孙海元^[23]在对生长育肥猪的研究中也得到类似的结论。本研究中,添加不同水平小肽没有使免疫球蛋白含量发生显著变化,对机体体液免疫无显著影响,与上述报道一致。

3.4 对育肥牦牛消化道PepT1 mRNA表达的影响

大量的研究表明,反刍动物消化道中PepT1的表达量存在物种差异性。在对奶牛、绵羊、湖羊的研究中发现,回肠、十二指肠和空肠的PepT1 mRNA表达量较高,在瓣胃和瘤胃中的表达量较低,在盲肠中最少^[12,14-16];但杂交肉牛中,瘤胃和瓣胃的PepT1 mRNA表达量要高于十二指肠,由低到高依次为盲肠、皱胃、网胃、十二指肠、瘤胃、瓣胃、空肠、回肠^[13];而在犊牛中,胃部的PepT1 mRNA表达量由低到高依次为皱胃、瓣胃、网胃、瘤胃^[27],与在奶牛和肉牛的研究上有所差异;在对放牧牦牛的研究中发现,PepT1 mRNA在小肠的表达量要高于胃部和大肠,由低到高依次为盲肠、皱胃、结肠、瓣胃、瘤胃、网胃、十二指肠、回肠、空肠,且网胃的表达量显著高于瘤胃、瓣胃和皱胃,但显著低于回肠和十二指肠^[28]。本试验是对舍饲育肥牦牛开展的研究,PepT1的组织表达特性与上述报道的放牧牦牛组织表达特性存在差异,在本研究中,PepT1 mRNA在小肠的表达量要高于前胃和真胃,由低到高依次为皱胃、瘤胃、瓣胃、网胃、十二指肠、回肠、空肠,但十二指肠、网胃、瓣胃、瘤胃的表达量无显著差异。造成上述差异的原因可能与品种、饲养方式以及饲料组成不同有关。另有研究表明,非肠系膜系统（瘤胃、网胃、瓣胃、皱胃、十二指肠）的小肽吸收量大于肠系膜系统（空肠、回肠、盲肠、结肠）^[29,30],而在本试验中,小肠段PepT1 mRNA的表达量在前胃和真胃之上,猜测原因可能是另外几种非耗能的转运方式占主导作用所致。有研究表明,小肽的转运方式主要包括：载体转运、渗透扩散、旁细胞穿透和胞吞4种方式^[10],而只有载体转运消耗能量。

有研究表明,消化道内底物的浓度会影响PepT1的表达活性,底物浓度较高时会增加转运载体的数量和活性^[31]。PepT1的转运依赖跨膜质子梯度以及胞内的pH,小肽和H⁺一同被PepT1转运进入细胞,而后再通过氢钠泵转出H^+[32]。在本研究中,随着日粮小肽添加水平的升高,瘤胃、网胃和空肠PepT1 mRNA的表达量线性增加,可能与消化道中底物和H⁺的浓度变化有关,消化道不同部位酸碱环境存在差异,而胞外酸度能够影响PepT1对肽的亲和力^[14],进而影响小肽的转运效率。

4 结论

日粮中添加小肽能够提高养分的表观消化率,提升育肥牦牛生产性能,而且能够改善肝脏功能,促进机体代谢,但对机体体液免疫无显著影响;PepT1 mRNA在育肥牦牛小肠的表达量要高于前胃和真胃,且瘤胃、网胃和空肠中PepT1 mRNA的表达量随小肽添加水平的升高而线性升高,促进了小肽的转运和吸收。本试验条件下,日粮中小肽添加水平为2.25%时饲喂效果最佳。

（责任编辑林鉴非）

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100只体重为(891.42±23.68)g,健康状况良好的断奶獭兔,随机分为5组.对照组为基础日粮组,A、B、C、D组分别为基础日粮添加0.25%、0.50%、0.75%、1.00%的小肽.试验预试期7 d,正试期30 d.试验结果表明:添加0.50%小肽后,獭兔平均日增重比对照组提高24.16%(P＜0.05),平均日采食量显著降低(P＜0.05),料重比降低25.19%(P＜0.05).添加小肽后,獭兔胸腺指数、下颌淋巴结重显著高于对照组(P＜0.05).獭兔日粮中添加小肽可以增强断奶獭兔生长性能和免疫性能.

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[D]. 泰安: 山东农业大学, 2007.

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[D]. Taian: Shandong Agricultural University, 2007. (in Chinese)

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Dietary proteins are cleaved within the intestinal lumen to oligopeptides which are further processed to small peptides (di- and tripeptides) and free amino acids. Although the transport of amino acids is mediated by several specific amino acid transporters, the proton-coupled uptake of the more than 8000 different di- and tripeptides is performed by the high-capacity/low-affinity peptide transporter isoform PEPT1 (SLC15A1). Its wide substrate tolerance also allows the transport of a repertoire of structurally closely related compounds and drugs, which explains their high oral bioavailability and brings PEPT1 into focus for medical and pharmaceutical approaches. Although the first evidence for the interplay of nutrient supply and PEPT1 expression and function was described over 20 years ago, many aspects of the molecular processes controlling its transcription and translation and modifying its transporter properties are still awaiting discovery. The present review summarizes the recent knowledge on the factors modulating PEPT1 expression and function in Caenorhabditis elegans, Danio rerio, Mus musculus and Homo sapiens, with focus on dietary ingredients, transcription factors and functional modulators, such as the sodium-proton exchanger NHE3 and selected scaffold proteins.

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李燕, 林雪彦, 姜运良, 苏鹏程, 王中华 . 应用RT-PCR方法检测牛消化道各段Ⅰ型肽载体mRNA差异表达的研究
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已有的研究表明，消化道可以小肽的形式吸收氨基酸，但尚未很好地确定小肽的主要吸收部位。我们在另一项研究中对牛Ⅰ型肽载体（BPepTⅠ）第3～10结构域1 566 bp片段序列进行了测定（Genebank登录号：DQ309694），与绵羊相同区域片段序列同源性高达96.04%。本研究以BPepⅠ测序结果为基础，并参考绵羊的部分序列设计了跨内含子引物，荧光实时定量聚合酶链反应（RTPCR）方法测定了牛离体各段消化道黏膜或上皮组织BPepTⅠ的相对表达水平，以评估小肽在牛消化道的主要吸收部位。以持家基因βactin为参比基因，结肠样品为校正样品（其相对表达量2-△△CT值为1），研究结果表明：在整段消化道中，BPepTⅠ的相对表达量（2-△△CT值）由高到低依次为回肠（30.62）、空肠（26.12）、瓣胃（22.61）、瘤胃（16.17）、十二指肠（11.97）、网胃（5.18）、皱胃（3.22）、盲肠（1.13）和结肠（1.00）。各段消化道BPepTⅠ相对表达量差异显著（P<0.05），其中回肠相对表达量与空肠和瓣胃差异不显著（P>0.05），与瘤胃、十二指肠、网胃、皱胃、盲肠、结肠差异显著（P<0.05）；空肠相对表达量与瓣胃、瘤胃、十二指肠差异不显著（P>0.05），与网胃、皱胃、盲肠、结肠差异显著（P<0.05）；网胃、皱胃、盲肠、结肠的2-△△CT值均在5.2以下，相互之间相对表达量的差异不显著（P>0.05）。由BPepTⅠ基因相对表达活性可以将牛的各段消化道分为3组，回肠、空肠、瓣胃的活性最高，其次为瘤胃和十二指肠，网胃、皱胃、盲肠、结肠则最低。

, LIN X

, JIANG Y

, SU P

, WANG Z

. A study on the relative expression of peptide transporterI mRNA in different parts of bovine gastrointestinal tract by RT-PCR
Chinese Journal of Animal Nutrition, 2008,20(2):183-190. (in Chinese)

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[D]. 杭州: 浙江大学, 2015.

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[D]. Hangzhou: Zhejiang University, 2015. (in Chinese)

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[D]. 杨凌: 西北农林科技大学, 2017.

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[D]. Yangling: Northwest Agriculture & Forestry University, 2017. (in Chinese)

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There is a need to standardize the NDF procedure. Procedures have varied because of the use of different amylases in attempts to remove starch interference. The original Bacillus subtilis enzyme Type IIIA (XIA) no longer is available and has been replaced by a less effective enzyme. For fiber work, a new enzyme has received AOAC approval and is rapidly displacing other amylases in analytical work. This enzyme is available from Sigma (Number A3306; Sigma Chemical Co., St. Louis, MO). The original publications for NDF and ADF (43, 53) and the Agricultural Handbook 379 (14) are obsolete and of historical interest only. Up to date procedures should be followed. Triethylene glycol has replaced 2-ethoxyethanol because of reported toxicity. Considerable development in regard to fiber methods has occurred over the past 5 yr because of a redefinition of dietary fiber for man and monogastric animals that includes lignin and all polysaccharides resistant to mammalian digestive enzymes. In addition to NDF, new improved methods for total dietary fiber and nonstarch polysaccharides including pectin and beta-glucans now are available. The latter are also of interest in rumen fermentation. Unlike starch, their fermentations are like that of cellulose but faster and yield no lactic acid. Physical and biological properties of carbohydrate fractions are more important than their intrinsic composition.

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程茂基 . 绵羊瘤胃内寡肽的产生、降解、吸收、流通与微生物摄取规律的研究
[D]. 呼和浩特: 内蒙古农业大学, 2000.

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CHENG M

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[D]. Huhehot: Inner Mongolia Agricultural University, 2000. (in Chinese)

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李琍, 丁角立 . 肽对体外混合培养瘤胃微生物发酵和生长影响的研究
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[D]. 邯郸: 河北工程大学, 2013.

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[D]. Handan: Hebei University of Engineering, 2013. (in Chinese)

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史晓雪, 牛占宇, 幸超, 张三润, 闫婉姝, 张润厚 . 饲粮中添加梧桐子油和罗格列酮对绵羊生产性能、血清激素和生化指标的影响
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Twenty wether lambs (46 +/- 2 kg) fitted with ruminal and abomasal infusion catheters were used in a completely randomized design to determine the effects of differing proportions of ruminal and abomasal casein infusion on N balance in lambs fed low-quality brome hay (0.8% N, DM basis) for ad libitum intake. Wethers were infused with 0 (control) or 10.7 g/d of N from casein with ratios of ruminal:abomasal infusion of 100:0 (100R:0A), 67:33 (67R:33A), 33:67 (33R:67A), or 0:100% (0R:100A), respectively, over a 12-d period. Total N supply (hay N intake + N from casein infusion) was greater (P = 0.001) in lambs receiving casein infusion than in controls. Urinary N excretion (g/d) was greater (P = 0.001) in lambs receiving casein infusion than in controls. Urinary N excretion decreased as casein infusion was shifted from 100R:0A to 33R:67A and then slightly increased in lambs receiving 0R:100A (quadratic, P = 0.02). Total N excretion was greater (P = 0.001) in lambs receiving casein infusion than in controls and decreased linearly (P = 0.005) as casein infusion was shifted to the abomasum. Retained N (g/d, % of N intake, and % of digested N) was greater (P = 0.001) in lambs receiving casein than in controls. Retained N increased as infusion was shifted from 100R:0A to 33R:67A and then slightly decreased in lambs receiving 0R: 100A (quadratic, P < 0.07). Based on regression analysis, the predicted optimum proportion of casein infusion to maximize N retention was 68% into the abomasum. The regression suggests that supplementation with undegradable intake protein had an additional benefit over supplementation with ruminally degradable intake protein (100R:0A) and that changing the percentage of ruminally undegradable intake protein in supplemental protein from 33 to 100% resulted in minimal differences in N retention. Apparent N, DM, OM, and energy digestibility (% of intake) was greater (P < 0.03) in lambs infused with casein than controls but did not differ among casein infusion groups. These data suggest that feeding protein supplements containing a portion (greater than 0%) of the crude protein as ruminally undegradable intake protein, as compared to 100% ruminally degradable intake protein, to lambs consuming low-quality forage increases N retention and the efficiency of N utilization without influencing total-tract nutrient digestion.

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黑龙江畜牧兽医, 2017(5):88-91.

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This experiment investigated the effect of intraruminal infusion of propionic acid on the net flux of nitrogenous compounds across the mesenteric- (MDV) and portal-(PDV) drained viscera of seven Friesian steers, average BW 127 kg (SEM 4.6), fed a grass-pellet diet. Each received by random allocation 0 (control), .5, or 1.0 mol of propionic acid/d for 7 d. Blood flow in mesenteric and portal veins was determined by downstream dilution of p-aminohippuric acid in order to determine net appearance rates across the gastrointestinal tissues. Net urea and ammonia flux was unaffected by propionic acid supply. Circulating plasma free amino acid concentrations were increased (P < .05) by propionic acid infusion (2,235, 2,428, and 2,427, error mean square [EMS] 44,370 microM, for control, .5, and 1.0 mol of propionic acid/d, respectively). Net amino acid flux rates were increased at the highest rate of propionic acid infusion across MDV and PDV (4.66, 3.69, and 6.11, EMS 2.98 mol/d for MDV [P < .05] and 2.98, 2.45, and 3.73, EMS 1.69 mol/d for PDV [P < .10] for control, .5, and 1.0 mol of propionic acid/d respectively). Positive venous-arterio concentration differences for peptide-bound amino acids (PBAA) across the MDV and PDV indicated net appearance across the gastrointestinal tissues, but this was not affected by propionic acid infusion. The data show that amino acid flux across postruminal tissues can be influenced by ruminal propionic acid supply and that this does not affect PBAA appearance.

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[D]. Yangling: Northwest Agriculture & Forestry University, 2017. (in Chinese)

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黄薪蓓, 许庆彪, 刘建新 . 肠道氨基酸和小肽转运载体的基因表达、影响因素与分子调控机制
动物营养学报, 2015,27(1):21-27.

DOI:10.3969/j.issn.1006-267x.2015.01.004 URL [本文引用: 1]

饲粮蛋白质在瘤胃或肠道被降解成多肽,并进一步被分解成氨基酸和小肽,然后被吸收、利用。肠道氨基酸的转运受多种氨基酸转运载体,如中性、酸性和碱性氨基酸转运载体等的调节,小肽的转运则由小肽转运载体1介导。目前,对氨基酸及小肽转运载体基因的表达和功能调节相关的分子机制还不清楚,有待于进一步研究。本文综述了动物小肠肽与氨基酸转运载体等,重点介绍了其基因表达调节的分子机制、影响因素以及营养调控方面的研究进展。

HUANG X

, XU Q

, LIU J

. Gene expression, influence factors and molecular regulation of amino acid and peptide transporters in intestine
Chinese Journal of Animal Nutrition, 2015,27(1):21-27. (in Chinese)

DOI:10.3969/j.issn.1006-267x.2015.01.004 URL [本文引用: 1]