Effect of Low Protein Diet Supplemented with Rumen Protected Methionine and Rumen Protected Lysine on Performance, Nutrient Digestibility and Nitrogen Metabolism of Lactating Dairy Cows

OUYANG Tong; ZHOU Zhiyuan; LONG Shenfei; LI Shengli

doi:10.12418/CJAN2025.554

2025 , Vol. 37 >Issue 10: 6796 - 6811

DOI: https://doi.org/10.12418/CJAN2025.554

RESEARCH PAPER

Effect of Low Protein Diet Supplemented with Rumen Protected Methionine and Rumen Protected Lysine on Performance, Nutrient Digestibility and Nitrogen Metabolism of Lactating Dairy Cows

OUYANG Tong ,
ZHOU Zhiyuan ,
LONG Shenfei ,
LI Shengli ^,^*

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State Key Laboratory of Animal Nutrition and Feeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

^*professor, E-mail: lisheng0677@163.com

Received date: 2025-03-21

Online published: 2025-10-15

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Abstract

This experiment aimed to investigate the effects of low protein diet supplemented with rumen protected methionine (RPMet) and rumen-protected lysine (RPLys) on performance, nutrient digestibility and nitrogen metabolism of lactating dairy cows using the updated NASEM (2021) model, with the goal of providing experimental evidence and data support for the promotion and application of low protein diets in dairy cows. Thirty-six healthy Holstein dairy cows with similar parity [(1.86±0.90) parities], days in milk [(101.56±41.22) d], and milk yield [(39.71±6.28) kg/d] were randomly divided into 3 groups with 12 cows per group. The adequate metabolizable protein group (AMP group) was fed a diet with a crude protein (CP) level of 16.5%, meeting 101% of the metabolizable protein (MP) requirement, the deficient metabolizable protein group (DMP group) was fed a diet with a CP level of 15.0%, meeting 92% of the MP requirement. The deficient metabolizable protein+amino acid group (DMP+AA group) was supplemented with 41 g/d RPMet and 61 g/d RPLys on the basis of the DMP diet. The pre-test period lasted for 10 days, and the formal test period was 60 days. The results showed as follows: 1) there was no significant difference in milk yield among the groups (P>0.05), while the milk urea nitrogen content in the AMP group was extremely significantly higher than that in the DMP and DMP+AA groups (P<0.01). 2) The total tract apparent digestibility of dry matter (DM) in the DMP+AA group was extremely significantly higher than that in the AMP group (P<0.01), the total tract apparent digestibility of ether extract (EE) in the DMP group was extremely significantly higher than that in the AMP group (P<0.01). 3) The ammonia nitrogen (NH₃-N) content in rumen fluid in the AMP group was significantly higher than that in the DMP and DMP+AA groups (P<0.05), and the total volatile fatty acid content was extremely significantly higher than that in the DMP and DMP+AA groups (P<0.01); the microbial protein (MCP) content in rumen fluid in the AMP and DMP+AA groups was extremely significantly higher than that in the DMP group (P<0.01). 4) Compared with the AMP group, the urine nitrogen and the percentage of urine nitrogen to total nitrogen excretion in the DMP and DMP+AA groups were extremely significantly reduced (P<0.01), and the total nitrogen excretion was significantly reduced (P<0.05); the nitrogen utilization rate in the DMP+AA group was significantly higher than that in the AMP group (P<0.05), with no significant difference compared to the DMP group (P>0.05). 5) The plasma methionine content in the DMP+AA group was significantly higher than that in the AMP group (P<0.05) and extremely significantly higher than that in the DMP group (P<0.01). In conclusion, supplementing RPMet and RPLys in low protein diet can optimize the amino acid balance in small intestine, numerically improve the milk yield of mid-lactation dairy cows, effectively reduce nitrogen excretion, especially the amount and proportion of urine nitrogen, and enhance nitrogen utilization efficiency.

Key words： lactating dairy cows; low protein diet; rumen protected amino acids; performance; nitrogen metabolism

Cite this article

OUYANG Tong , ZHOU Zhiyuan , LONG Shenfei , LI Shengli . Effect of Low Protein Diet Supplemented with Rumen Protected Methionine and Rumen Protected Lysine on Performance, Nutrient Digestibility and Nitrogen Metabolism of Lactating Dairy Cows[J]. Chinese Journal of Animal Nutrition, 2025 , 37(10) : 6796 -6811 . DOI: 10.12418/CJAN2025.554

近年来,我国奶牛养殖业规模化程度不断提升,国家统计局数据显示,2024年我国牛奶产量已达4 079万t^[1]。奶牛的氮利用率偏低,氮排泄量较大,随着奶业的迅速发展,大量排泄氮对环境造成的污染问题也日益凸显。同时,大型规模化牧场为追求高产奶量,往往将饲粮蛋白质水平设置得较高,而过量饲喂蛋白质容易造成氮损失和氮资源的浪费。更值得关注的是,我国的蛋白质饲料原料长期依赖进口,其中大豆的进口依存度超过85%^[2]。国际市场豆粕价格的持续走高且波动不定,进一步推动了养殖业生产成本的上涨,给行业发展带来了诸多困扰。为此,农业农村部从2021年起制定并实施饲用豆粕减量替代方案,除了开发本地优质蛋白质饲料原料、优化饲粮结构外,还大力推行低蛋白质饲粮氨基酸平衡技术,重视饲粮的精准配方和精细加工。在泌乳奶牛的饲养中,低蛋白质饲粮是指在借鉴发达国家奶牛饲养标准的基础上,将饲粮粗蛋白质(CP)水平降至16%以下,同时通过添加过瘤胃氨基酸(rumen protected amino acid,RPAA),充分平衡奶牛饲粮中的必需氨基酸,从而维持甚至提高奶牛产奶量、乳蛋白产量和氮利用率,减少氮排泄及饲粮氮损失对环境的污染^[3]。目前,已有大量试验依据NRC(2001)等奶牛营养需要体系研究了低蛋白质饲粮中添加1种或多种RPAA对奶牛的影响,这些研究主要关注奶牛小肠可代谢蛋白(metabolizable protein,MP)、瘤胃可降解蛋白(rumen degradable protein,RDP)需要量,以及饲粮提供的蛋氨酸和赖氨酸占MP的比例^[4-6]。2021年,美国NASEM发布了最新版《奶牛营养需要》^[7],其通过考虑单个必需氨基酸供应量和能量供应对乳蛋白产量的影响,更新了可变化的MP利用效率,设置了单个必需氨基酸的目标利用率,对奶牛的蛋白质和氨基酸供应及推荐需要量提出了新的估算方法。但就目前而言,在我国奶牛养殖业产奶量高(35~40 kg/d)、饲粮CP水平高的背景下,应用这一新模型对低蛋白质氨基酸平衡饲粮进行评估的研究仍然较少。因此,本试验旨在利用新版NASEM(2021)模型,探究低蛋白质饲粮补充过瘤胃蛋氨酸(rumen protected methionine,RPMet)和过瘤胃赖氨酸(rumen protected lysine,RPLys)对泌乳奶牛生产性能、营养物质消化率和氮代谢的影响,分析奶牛在低蛋白质氨基酸平衡饲粮条件下能否维持较高生产性能、提高氮利用率并减少氮损失。通过评估奶牛低蛋白质饲粮的可行性,响应国家饲用豆粕减量替代的政策号召,为其推广应用提供试验依据和数据支持。

1 材料与方法

1.1 试验材料

试验所用RPMet和RPLys均为市售产品,RPMet含有57%蛋氨酸羟基类似物(HMBi),HMBi的蛋氨酸当量为78%,其中50%通过瘤胃壁吸收,可代谢蛋氨酸(dMet)含量为222 g/kg;RPMet中赖氨酸含量为40%,过瘤胃率为80%,小肠释放率为80%,可代谢赖氨酸(dLys)含量为256 g/kg。

1.2 试验设计与饲粮

本试验方案已通过中国农业大学实验动物福利与动物实验伦理审查委员会审查,批准编号:AW70405202-1-01。试验选取36头胎次[(1.86±0.90)胎]、泌乳天数[(101.56±41.22) d]和产奶量[(39.71±6.28) kg/d]相近的健康荷斯坦奶牛,随机分为3组,每组12头。可代谢蛋白充足组(AMP组)饲喂CP水平为16.5%,满足101% MP需要量的饲粮,可代谢蛋白不足组(DMP组)饲喂CP水平为15.0%,满足92% MP需要量的饲粮,可代谢蛋白不足+氨基酸组(DMP+AA组)在DMP饲粮基础上补充41 g/d RPMet和61 g/d RPLys。预试期10 d,正试期60 d。

参照NASEM(2021)^[7]使用NASEM Dairy-8软件配制试验饲粮,确保除CP外各项营养指标均满足41月龄、泌乳天数94 d、2胎、非妊娠、体重700 kg、产奶量40 kg/d、乳脂率3.95%、乳真蛋白率3.00%、乳蛋白率3.23%、乳糖率4.85%及体况评分3.0的荷斯坦奶牛的营养需要。部分饲料原料的营养物质含量见表1,氨基酸含量见表2;试验饲粮组成及营养水平见表3,蛋白质与氨基酸平衡情况见表4。

表1 饲料原料营养物质含量(干物质基础)

Table 1 Nutrient content of feed ingredients (DM basis) %

项目 Items	干物质 DM	粗蛋白质 CP	粗脂肪 EE	粗灰分 Ash	中性洗涤纤维 NDF	酸性洗涤纤维 ADF
豆粕Soybean meal	88.85	48.52	1.31	6.99	10.56	7.16
膨化大豆Extruded soybean	91.78	37.03	21.22	5.58	10.38	7.10
苔草Sedge	95.02	9.52	1.84	6.45	58.25	32.73
苜蓿干草Alfalfa hay	95.12	16.12	1.45	8.55	49.38	35.60
酵母培养物Yeast culture	93.03	23.10	7.60	19.23	25.67	10.28
双低菜籽粕Double-low rapeseed meal	91.82	41.72	3.42	7.28	30.02	19.64
压片玉米Steam-flaked corn	87.57	7.25	2.47	0.96	7.66	1.96
大麦麸皮Barley bran	91.89	7.81	2.68	11.22	53.50	23.18
玉米Corn	86.92	9.39	3.93	1.58	7.42	1.72
湿贮玉米High moisture corn	53.80	9.54	4.55	1.73	14.45	7.54
玉米青贮Corn silage	33.21	7.27	3.71	4.24	36.68	20.89
全棉籽Whole cottonseed	93.73	25.89	15.89	4.44	45.44	36.98

干物质为风干基础。DM was as air-dry basis.

表2 饲料原料氨基酸含量(粗蛋白质基础)

Table 2 Amino acid content of feed ingredients (CP basis) %

项目 Items	豆粕 Soybean meal	膨化大豆 Extruded soybean	双低菜籽粕 Double- low rapeseed meal	全棉籽 Whole cotton- seed	玉米青贮 Corn silage	玉米 Corn	湿贮玉米 High moisture corn	压片玉米 Steam- flaked corn	苜蓿干草 Alfalfa hay	苔草 Sedge	酵母培养物 Yeast culture	大麦麸皮 Barley bran
必需氨基酸EAA
精氨酸Arg	7.12	6.92	5.23	11.61	1.75	5.02	4.50	4.03	2.58	4.63	3.76	4.80
组氨酸His	2.64	2.58	2.42	2.72	1.62	2.94	2.63	3.03	1.53	1.75	2.18	1.98
异亮氨酸Ile	4.58	4.39	3.56	2.98	3.50	3.42	3.06	3.59	2.80	4.39	3.51	3.47
亮氨酸Leu	7.80	7.57	6.50	5.68	9.14	12.55	11.23	13.98	4.68	8.45	8.73	6.75
赖氨酸Lys	6.68	6.55	5.50	4.51	3.00	3.39	3.04	2.79	4.28	5.93	3.96	5.06
蛋氨酸Met	1.23	1.31	1.70	1.42	1.82	2.55	2.18	2.41	0.83	1.73	1.55	1.62
苯丙氨酸Phe	5.09	4.89	3.72	5.20	4.31	5.21	4.66	5.45	3.09	5.36	4.22	4.39
色氨酸Trp	4.06	4.00	4.06	3.22	3.44	3.77	3.38	3.81	3.12	4.60	3.65	1.04
苏氨酸Thr	1.65	1.58	1.13	1.57	0.39	0.95	0.54	0.81	1.06	1.70	0.95	3.92
缬氨酸Val	4.70	4.54	4.62	4.17	4.79	4.84	4.33	4.95	3.68	5.18	4.66	4.75
非必需氨基酸NEAA
丙氨酸Ala	4.39	4.32	4.15	3.79	8.43	7.74	6.93	8.27	3.56	5.82	6.47	5.31
天冬氨酸Asp	11.42	11.05	6.49	9.39	6.98	7.18	6.43	6.84	11.04	9.48	8.04	8.03
半胱氨酸Cys	1.46	1.42	2.24	1.69	1.46	2.45	1.91	2.52	1.00	1.26	1.62	1.84
谷氨酸Glu	18.81	18.01	16.52	19.92	11.47	19.81	17.73	20.61	6.43	10.69	15.12	15.98
甘氨酸Gly	4.29	4.23	4.95	3.96	4.13	4.06	3.63	3.75	3.07	5.13	3.88	4.75
脯氨酸Pro	5.17	4.98	5.86	3.67	6.70	9.20	8.23	9.63	5.95	4.90	6.01	7.07
丝氨酸Ser	5.22	5.09	3.99	4.34	3.49	4.90	4.39	5.10	3.76	4.67	4.11	4.25
酪氨酸Tyr	3.25	3.26	2.33	2.67	2.66	3.39	3.04	3.43	1.81	3.04	3.42	2.61

表3 试验饲粮组成及营养水平(干物质基础)

Table 3 Composition and nutrient levels of experimental diets (DM basis) %

项目 Items	组别Groups
项目 Items	AMP	DMP	DMP+AA
原料Ingredients
玉米青贮Corn silage	32.76	32.76	32.76
玉米Corn	13.52	15.17	15.17
豆粕Soybean meal	13.32	11.63	11.63
压片玉米Steam-flaked corn	8.31	8.31	8.31
苜蓿干草Alfalfa hay	7.22	7.22	7.22
苔草Sedge	4.33	4.33	4.33
双低菜籽粕Double-low rapeseed meal	3.83	3.14	3.14
全棉籽Whole cottonseed	3.56	4.98	4.98
甘蔗糖蜜Cane molasses	3.47	3.47	3.47
膨化大豆Extruded soybean	3.13
湿贮玉米High moisture corn	2.04	2.04	2.04
预混料Premix¹⁾	1.71	1.71	1.71
小苏打NaHCO₃	0.83	0.83	0.83
碳酸钙CaCO₃	0.57	0.57	0.57
棕榈脂肪粉Palm fat powder	0.57	0.57	0.57
酵母培养物Yeast culture	0.53	0.53	0.53
氧化镁MgO	0.16	0.16	0.16
食盐NaCl	0.11	0.11	0.11
脱霉剂Mycotoxin binder	0.03	0.03	0.03
大麦麸皮Barley bran		2.44	2.44
合计Total	100.00	100.00	100.00
营养水平Nutrient levels²⁾
粗蛋白质CP	16.51	14.95	15.05
产奶净能NE_L/(MJ/kg)	7.36	7.23	7.23
粗脂肪EE	4.40	4.05	4.14
淀粉Starch	27.13	28.54	28.45
粗灰分Ash	8.04	8.06	8.12
中性洗涤纤维NDF	25.76	27.10	26.99
酸性洗涤纤维ADF	16.23	16.95	16.89
可发酵碳水化合物Ferm. CHO	45.94	46.52	46.34
非纤维碳水化合物NFC	45.29	45.84	45.69
钙Ca	0.71	0.70	0.70
总磷TP	0.38	0.39	0.39
额外补充Extra supplements
过瘤胃蛋氨酸RPMet/(g/d)			41
过瘤胃赖氨酸RPLys/(g/d)			61

1)每千克预混料提供 One kilogram of premix provided:VA 220 000 IU,VD₃ 72 000 IU,VE 2 000 IU,D-生物素 D-biotin 40 mg,烟酰胺 nicotinamide 2 000 mg,Fe (as ferrous sulfate) 830 mg,Cu (as copper chloride) 680 mg,Mn (as manganese sulfate) 710 mg,Zn (as zinc sulfate) 2 005 mg,Mg (as magnesium oxide) 33 000 mg,Co (as cobaltous sulfate) 12 mg。2)产奶净能、可发酵碳水化合物、非纤维碳水化合物为根据试验牛实际生产数据(干物质采食量、泌乳天数、体重、产奶量、乳成分等)及饲料原料营养物质含量(表1),使用NASEM(2021)模型所得计算值,其余为实测值。NE_L, Ferm. CHO and NFC were calculated values derived using the NASEM (2021) model, based on the actual production data of experimental cows (including dry matter intake, days in milk, body weight, milk yield, milk composition, etc.) and the nutrient content of feed ingredients (Table 1), while the others were measured values.

表4 试验饲粮蛋白质与氨基酸平衡情况

Table 4 Protein and amino acid balance of experimental diets

项目 Items	组别Groups
项目 Items	AMP	DMP	DMP+AA
蛋白质平衡Protein balance
可代谢蛋白需要量MP requirements/(g/d)	2 470	2 475	2 476
可代谢蛋白供应量MP supply/(g/d)	2 499	2 282	2 313
可代谢蛋白平衡MP balance/(g/d)	+29	-193	-163
瘤胃可降解蛋白供应量RDP supply/(% DM)	11.16	10.14	10.10
瘤胃非降解蛋白供应量RUP supply/(% DM)	5.33	4.79	4.90
瘤胃可降解蛋白:瘤胃非降解蛋白RDP:RUP	2.09	2.12	2.06
氨基酸平衡Amino acid balance
可代谢蛋氨酸dMet
需要量Requirements/(g/d)	60	60	60
饲粮供应量Supplied by diets/(g/d)	55	51	51
过瘤胃蛋氨酸供应量Supplied by RPMet/(g/d)			9
可代谢蛋氨酸平衡dMet balance/(g/d)	-5	-9	0
可代谢赖氨酸dLys
需要量Requirements/(g/d)	188	189	189
饲粮供应量Supplied by diets/(g/d)	191	173	173
过瘤胃赖氨酸供应量Supplied by RPLys/(g/d)			16
可代谢赖氨酸平衡dLys balance/(g/d)	+2	-16	0

	表中数据为根据试验牛实际生产数据(干物质采食量、泌乳天数、体重、产奶量、乳成分等)以及饲料原料营养物质(表1)、氨基酸含量(表2),使用NASEM(2021)模型所得计算值。
	Data in the table were calculated values derived using the NASEM (2021) model, based on the actual production data of experimental cows (including dry matter intake, days in milk, body weight, milk yield and milk composition) as well as the nutrient contents (Table 1) and amino acid contents (Table 2) of feed ingredients.

RPMet和RPLys补充量的计算方法:通过NASEM(2021)模型预测DMP组饲粮中dMet和dLys的供应量与奶牛需要量的差值(dMet -9 g/d,dLys -16 g/d),该差值即为DMP+AA组需通过RPMet和RPLys补充的量。根据RPMet和RPLys产品的dMet和dLys含量,确定RPMet补充量为41 g/d,RPLys补充量为61 g/d。

1.3 饲养管理

动物试验于河南省新乡市原阳县澳美牧业有限公司牧场开展。3组奶牛分别饲养在相邻且独立建造的牛舍内,各组饲养管理及环境条件保持一致,均采用散栏饲养模式。饲粮以全混合日粮(TMR)形式投放,其中RPMet和RPLys先与精料混合,再经搅拌机进一步搅拌制成TMR后进行饲喂。试验期间,奶牛全天自由饮水,每日挤奶3次(04:50、12:20和18:20),投料2次(05:00和18:30),2次投料量比例为49∶51,剩料量控制在5%~10%。

1.4 样品采集与制备

1.4.1 饲料原料及饲粮样品

试验开始前,采集饲料原料样品,用于测定营养物质含量以制作配方;试验期间,采用多点采样法,每周采集1次新鲜TMR及剩料样品,每组各样品采集量为500 g。样品于65 ℃恒温干燥箱中烘48 h后,在室温下回潮24 h,测定初水分含量,随后装入干燥密封袋,低温保存备用。

1.4.2 牛奶样品

每周连续3 d人工记录产奶量,将早、中、晚3次产奶量相加得到日产奶量,以连续3 d日产奶量的平均值作为周产奶量。正试期间,每2周(即第1、15、29、43和57天)采集1次奶样,按早、中、晚=4∶3∶3的比例混合,每头牛采集50 mL奶样,加入重铬酸钾防腐剂,于-4 ℃保存。

1.4.3 血液样品

在采集奶样的次日,即正试期第2、16、30、44和58天晨饲前,通过尾根静脉采集10 mL血液至乙二胺四乙酸(EDTA)抗凝管中。静置2 h后,以2 146×g的离心力离心15 min,吸取上清液转移至2 mL冻存管中,于-20 ℃保存备用。

1.4.4 瘤胃液样品

采血当天,每组随机选取8头奶牛,在晨饲后4 h用瘤胃导管采集100 mL瘤胃液,经4层纱布过滤后,立即使用手持便携式pH计测定瘤胃液pH,随后分装至15 mL离心管中,于-20 ℃保存备用。

1.4.5 粪便、尿液样品

在正试期最后3 d的8个时间点(第58天09:00、18:00和21:00,第59天12:00和15:00,第60天00:00、03:00和06:00),选取已采集瘤胃液的8头奶牛,于各时间点采用直肠取粪法收集每头牛100 g粪便样品,加入25 mL 10%酒石酸混匀,置于-20 ℃保存。采集粪便样品的同时,采用阴门按摩刺激法采集中段尿液,每头牛各时间点采集15 mL,迅速加入1 mol/L H₂SO₄(尿液与酸的比例为9∶1),置于-20 ℃保存。8个时间点采样结束后,将每头牛的所有粪样、尿样分别等量混合均匀。

1.5 指标检测

1.5.1 生产性能

试验期间,每天记录各组奶牛的TMR饲喂量和剩料量,并据此计算群体平均干物质采食量。每3周用体重秤测定1次体重(连续测定3 d取平均值)。

采集的奶样送至山西省奶牛生产性能测定管理中心,使用CombiFoss FT+乳成分及体细胞分析仪(Foss Analytical,丹麦)测定乳蛋白率、乳真蛋白率、乳脂率、乳糖率、乳尿素氮和全固形物含量以及乳中的体细胞数,并计算能量校正乳(ECM)产量,计算公式^[8]如下:

ECM产量(kg/d)=产奶量×[(38.3×乳脂率×10+24.2×乳真蛋白率×10+16.54×乳糖率×10+20.7)/3 140]。

1.5.2 营养物质全肠道表观消化率

饲料原料、饲粮与粪便样品经粉碎机粉碎后过1 mm筛,参照GB 6435—2014的方法测定干物质(DM)含量;参照GB/T 6432—2018的方法,使用KT-200快速定氮仪(Foss Analytical,丹麦)测定CP含量;参照GB/T 6433—2006的方法,使用XT-15i脂肪分析仪(ANKOM Technology,美国)测定粗脂肪(EE)含量;参照Van Soest等^[9]的方法,使用ANKOM-2000全自动纤维分析仪(ANKOM Technology,美国)测定中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量。饲粮钙(Ca)和总磷(TP)含量分别参照GB/T 6436—2018和GB/T 6437—2018的方法测定,淀粉含量采用Megazyme淀粉总量检测试剂盒(K-TSTA,爱尔兰)进行测定;饲粮和饲料原料粗灰分(Ash)含量参照GB/T 6438—2007的方法测定;饲料原料氨基酸含量参照GB/T 18246—2019的方法测定;饲粮和粪便盐酸不溶灰分(AIA)含量参照GB/T 23742—2009,采用灼烧和酸处理法测定。

以AIA为内源指示剂,计算营养物质的全肠道表观消化率(apparent total tract digestibility,ATTD),计算公式^[10]为:

ATTD(%)=[1-(Nf×Ad)/(Af×Nd)]×100。

式中:ATTD是某营养物质的全肠道表观消化率(%);Nf是粪便中该营养物质的含量(g/kg);Ad是饲粮中AIA的含量(g/kg);Af是粪便中AIA的含量(g/kg);Nd是饲粮中该营养物质的含量(g/kg)。

1.5.3 瘤胃发酵参数

将瘤胃液解冻后,采用苯酚-次氯酸钠比色法^[11]测定氨态氮(NH₃-N)含量,采用考马斯亮蓝比色法^[12]测定微生物蛋白(MCP)含量。解冻后的瘤胃液经预处理后,使用配备毛细管色谱柱的Agilent-6890N气相色谱仪(安捷伦科技有限公司,美国)测定挥发性脂肪酸(VFA)含量^[13]。每个样品设置3个重复。

1.5.4 氮代谢

采用酶联比色法,使用日立7020全自动生化分析仪测定血浆尿素氮(plasma urea nitrogen,PUN)含量。尿液中氮含量采用凯氏定氮法测定;尿素氮含量采用脲酶-谷氨酸脱氢酶偶联法测定;肌酐含量采用碱性苦味酸比色法,使用彩虹-722分光光度计进行测定;尿酸含量采用尿酸酶-过氧化物酶法测定。

日排尿量以肌酐为尿液标记物,参照Lee等^[14]的方法进行估算,假设肌酐排泄率恒定为27.3 mg/kg BW,计算公式为:

V=(BW×Y)/X。

式中:V为日排尿量(L/d);BW为体重(试验最后1周的体重);Y为肌酐排泄率27.3 mg/kg BW;X为尿液中肌酐含量(mg/L)。

氮代谢相关指标计算公式如下:

摄入氮(g/d)=干物质采食量×饲粮CP含量/6.25;

乳真蛋白氮(g/d)=产奶量×乳真蛋白率/6.38;

尿氮(g/d)=尿液中氮含量×日排尿量;

日排粪量(g/d)=(干物质采食量×饲粮AIA含量)/(粪便AIA含量×粪便DM含量);

粪氮(g/d)=粪便CP含量×日排粪量/6.25;

尿尿素氮(g/d)=尿液中尿素氮含量×日排尿量;

尿酸排泄量(mmol/d)=尿液中尿酸含量×日排尿量;

氮利用率(%)=(乳真蛋白氮/摄入氮)×100。

1.5.5 血浆游离脂肪酸含量

采用液相色谱串联质谱法测定血浆中18种游离氨基酸含量。

1.6 数据统计与分析

所有试验牛的周产奶量采用每周连续3 d测定值的平均值,共收集9周的重复测量数据;牛奶、瘤胃液和血液样品每2周采集1次,共获得5周的重复测量数据;粪便和尿液样品于试验最后3 d中的多个时间点采集并混合,每头牛1个样品,获得每组8头奶牛的生物学重复数据。重复测量数据采用SAS 9.4软件的MIXED模型进行统计分析,模型中固定效应包含处理、时间及二者的互作效应,随机效应为奶牛个体。在剔除异常值并完成正态性检验后,进行混合效应分析,采用Tukey法进行多重比较。对于粪便和尿液指标,因无重复测量数据,仅存在组内生物学重复,因此采用单因素方差分析(one-way ANOVA),模型中固定效应为处理,随机效应为奶牛个体,固定效应显著时采用Tukey法进行多重比较。所有数据结果均以最小二乘均值和均值标准误(SEM)表示,以P<0.05为差异显著,P<0.01为差异极显著。

2 结果

2.1 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛生产性能的影响

由表5可知,各组间产奶量差异不显著(P>0.05),从数值上看,AMP组产奶量最高,DMP+AA组与之接近,DMP组最低。乳尿素氮含量也以AMP组为最高,且极显著高于DMP和DMP+AA组(P<0.01)。

表5 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛生产性能的影响

Table 5 Effect of low protein diet supplemented with RPMet and RPLys on performance of lactating dairy cows

项目 Items	组别Groups			SEM	P值P-value
项目 Items	AMP	DMP	DMP+AA	SEM	处理 Treatment	时间 Time	处理×时间 Treatment×time
体重BW/kg	655.33	645.22	636.74	22.917	0.848	0.526	0.425
干物质采食量DMI/(kg/d)	24.37	25.31	24.69
产奶量Milk yield/(kg/d)	36.49	35.83	36.25	1.791	0.965	<0.001	0.867
能量校正乳产量ECM yield/(kg/d)	34.27	33.36	32.88	1.428	0.750	<0.001	0.693
乳真蛋白率Milk true protein rate/%	3.08	3.03	3.13	0.079	0.708	<0.001	0.957
乳蛋白率Milk protein rate/%	3.28	3.22	3.32	0.078	0.638	<0.001	0.928
乳蛋白产量Milk protein yield/(kg/d)	1.18	1.12	1.19	0.053	0.610	0.094	0.839
乳脂率Milk fat rate/%	3.60	3.66	3.39	0.274	0.763	<0.001	0.243
乳脂产量Milk fat yield/(kg/d)	1.29	1.30	1.18	0.086	0.514	<0.001	0.429
乳糖率Lactose rate/%	5.23	5.10	5.18	0.045	0.130	<0.001	<0.001
乳糖产量Lactose yield/(kg/d)	1.90	1.77	1.87	0.094	0.590	<0.001	0.075
乳尿素氮含量MUN content/(mg/dL)	17.02^Aa	14.88^Bb	15.35^Bb	0.480	<0.001	<0.001	<0.001
体细胞数SCC/(×10³个/mL)	54.3	81.0	98.4	19.37	0.280	0.850	0.228
全固形物含量TS content/%	12.11	12.47	12.33	0.310	0.719	<0.001	0.287

	同行数据肩标无字母或相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05),不同大写字母表示差异极显著(P<0.01)。下表同。
	In the same row, values with no letter or the same small letter superscripts mean no significant difference (P>0.05), while with different small letter superscripts mean significant difference (P<0.05), and with different capital letter superscripts mean extremely significant difference (P<0.01). The same as below.

2.2 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛营养物质全肠道表观消化率的影响

由表6可知,DMP+AA组DM全肠道表观消化率极显著高于AMP组(P<0.01),而与DMP组无显著差异(P>0.05);3组间CP、NDF和ADF全肠道表观消化率差异均不显著(P>0.05);DMP组EE全肠道表观消化率最高,极显著高于AMP组(P<0.01),与DMP+AA组差异不显著(P>0.05)。

表6 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛营养物质全肠道表观消化率的影响

Table 6 Effect of low protein diet supplemented with RPMet and RPLys on nutrient apparent total tract digestibility of lactating dairy cows %

项目 Items	组别Groups			SEM	P值 P-value
项目 Items	AMP	DMP	DMP+AA	SEM	P值 P-value
干物质DM	70.97^Bb	72.68^AaBb	76.57^Aa	1.158	<0.001
粗蛋白质CP	72.48	71.02	70.95	1.132	0.563
中性洗涤纤维NDF	51.95	49.08	55.13	2.226	0.113
酸性洗涤纤维ADF	51.77	49.12	53.09	1.961	0.352
粗脂肪EE	65.22^Bb	76.16^Aa	73.79^Aa	2.317	<0.001

2.3 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛瘤胃发酵参数的影响

由表7可知,AMP组瘤胃液NH₃-N含量显著高于DMP和DMP+AA组(P<0.05),AMP和DMP+AA组瘤胃液MCP含量均极显著高于DMP组(P<0.01);AMP组瘤胃液丙酸、异戊酸含量显著高于DMP组(P<0.05),总挥发性脂肪酸含量极显著高于DMP组(P<0.01);DMP+AA组瘤胃液丙酸、异戊酸和戊酸含量与AMP组均无显著差异(P>0.05),总挥发性脂肪酸含量极显著低于AMP组(P<0.01),与DMP组差异不显著(P>0.05)。

表7 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛瘤胃发酵参数的影响

Table 7 Effect of low protein diet supplemented with RPMet and RPLys on rumen fermentation parameters of lactating dairy cows

项目 Items	组别Groups			SEM	P值P-value
项目 Items	AMP	DMP	DMP+AA	SEM	处理 Treatment	时间 Time	处理×时间 Treatment×time
pH	6.12	6.27	6.20	0.045	0.065	<0.001	<0.001
氨态氮NH₃-N/(mg/dL)	33.74^a	28.21^b	26.42^b	1.875	0.022	<0.001	0.499
微生物蛋白MCP/(mg/mL)	89.12^Aa	74.81^Bb	86.10^Aa	3.149	<0.001	<0.001	0.133
乙酸Acetate/(mmol/L)	47.99	44.52	44.37	1.451	0.137	<0.001	<0.001
丙酸Propionate/(mmol/L)	24.40^a	20.23^b	23.29^a	1.076	0.020	<0.001	<0.001
异丁酸Isobutyrate/(mmol/L)	0.77	0.72	0.75	0.028	0.337	<0.001	<0.001
丁酸Butyrate/(mmol/L)	12.38	11.48	11.81	0.493	0.438	<0.001	<0.001
异戊酸Isovalerate/(mmol/L)	1.57^a	1.27^b	1.40^ab	0.068	0.012	<0.001	0.027
戊酸Valerate/(mmol/L)	1.74^ab	1.58^b	1.99^a	0.105	0.032	<0.001	<0.001
总挥发性脂肪酸TVFA/(mmol/L)	96.34^Aa	82.68^Bb	86.19^Bb	2.720	<0.001	<0.001	<0.001
乙酸/丙酸Acetate/propionate	1.96	2.08	1.96	0.078	0.396	<0.001	0.220

2.4 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛氮代谢的影响

由表8可知,与AMP组相比,DMP和DMP+AA组尿氮及尿氮占总排出氮比例极显著降低(P<0.01),总排出氮显著降低(P<0.05)。AMP组血浆尿素氮含量显著高于DMP组(P<0.05),而与DMP+AA组差异不显著(P>0.05)。DMP+AA组氮利用率显著高于AMP组(P<0.05),与DMP组差异不显著(P>0.05)。

表8 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛氮代谢的影响

Table 8 Effect of low protein diet supplemented with RPMet and RPLys on nitrogen metabolism of lactating dairy cows

项目 Items	组别Groups			SEM	P值 P-value
项目 Items	AMP	DMP	DMP+AA	SEM	P值 P-value
摄入氮N intake/(g/d)	643.8	605.4	594.5
排出氮N excretion/(g/d)
乳真蛋白氮Milk TPN	168.8	167.8	176.2	7.65	0.662
尿氮Urine N	170.4^Aa	125.8^Bb	130.6^Bb	8.91	<0.001
尿尿素氮UUN	71.9	63.1	58.5	5.47	0.239
粪氮Fecal N	185.7	171.5	169.3	7.09	0.230
总排出氮Total N excretion	525.0^a	466.0^b	473.0^b	10.60	0.021
尿尿素氮/尿氮UUN/Urine N/%	42.4	48.6	45.0	4.22	0.583
占总排出氮比例Percentage of total N excretion/%
粪氮Fecal N	35.5	36.4	35.8	1.67	0.911
尿氮Urine N	32.9^Aa	26.9^Bb	27.2^Bb	1.55	0.008
占摄入氮比例Percentage of N intake/%
粪氮Fecal N	27.5	29.0	29.1	1.13	0.563
尿氮Urine N	26.5^a	20.8^b	22.0^ab	1.46	0.022
总排出氮Total N excretion	81.5	76.9	79.6	2.19	0.636
氮利用率NUE/%	25.8^b	27.9^ab	31.2^a	1.67	0.030
日排尿量Daily urine output/(L/d)	45.5	42.0	46.2	8.19	0.929
尿酸排泄量Uric acid output /(mmol/d)	58.9	63.8	53.2	4.16	0.217
血浆尿素氮含量PUN content/(mg/dL)	5.10^a	3.99^b	4.57^ab	0.303	0.046

2.5 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛血浆游离氨基酸含量的影响

由表9可知,DMP+AA组血浆蛋氨酸含量显著高于AMP组(P<0.05),极显著高于DMP组(P<0.01),AMP组血浆蛋氨酸含量极显著高于DMP组(P<0.01)。

表9 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛血浆游离氨基酸含量的影响

Table 9 Effect of low protein diet supplemented with RPMet and RPLys on plasma free amino acid content of lactating dairy cows μmol/L

项目 Items	组别Groups			SEM	P值P-value
项目 Items	AMP	DMP	DMP+AA	SEM	处理 Treatment	时间 Time	处理×时间 Treatment×time
必需氨基酸EAA
精氨酸Arg	13.60	12.98	14.70	0.772	0.300	0.083	0.485
组氨酸His	26.94	25.37	27.03	1.493	0.675	<0.001	0.785
异亮氨酸Ile	86.08	86.56	87.96	3.893	0.939	<0.001	0.285
亮氨酸Leu	138.53	138.90	146.32	5.229	0.505	0.241	0.065
赖氨酸Lys	16.47	15.33	17.13	0.727	0.235	<0.001	0.153
蛋氨酸Met	44.59^Ab	38.31^Bc	50.60^Aa	1.940	<0.001	<0.001	0.255
苯丙氨酸Phe	52.09	51.34	48.42	2.364	0.520	0.306	0.220
色氨酸Trp	46.68	45.97	44.93	2.748	0.903	0.033	0.541
苏氨酸Thr	52.18	53.90	49.36	3.244	0.613	0.037	0.431
缬氨酸Val	186.97	188.50	191.55	6.653	0.885	0.527	0.044
总必需氨基酸Total EAA	664.13	657.18	677.67	20.400	0.772	0.416	0.100
非必需氨基酸NEAA
丙氨酸Ala	180.65	167.41	176.98	12.526	0.745	<0.001	0.216
天冬氨酸Asp	6.45	6.42	6.29	0.303	0.927	0.303	0.819
半胱氨酸Cys	1.16	1.19	1.11	0.108	0.848	0.833	0.413
谷氨酸Glu	17.96	18.50	15.97	1.016	0.205	0.010	0.788
甘氨酸Gly	144.70	132.19	139.38	8.076	0.556	<0.001	0.384
脯氨酸Pro	78.80	71.26	72.31	4.550	0.460	<0.001	0.761
丝氨酸Ser	44.45	42.18	40.19	2.081	0.367	<0.001	0.231
酪氨酸Tyr	66.34	62.45	66.08	2.950	0.590	<0.001	0.091

3 讨论

3.1 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛生产性能的影响

产奶量和乳蛋白产量是衡量奶牛生产性能的关键指标。已有大量研究报道了饲粮CP水平对奶牛产奶量和乳蛋白产量等生产指标的影响,其中部分研究显示,饲粮CP水平下降对奶牛泌乳性能的影响不显著^[15-16];但多数研究表明,饲粮CP水平降低会导致奶牛产奶量和乳蛋白产量显著下降^[17-18]。大量试验证实,在低蛋白质饲粮中补充RPAA,能够提高奶牛的产奶量及乳蛋白产量^[4]。Lee等^[19]将高产奶牛饲粮的CP水平从15.7%降至13.6%(MP缺乏15%),导致奶牛产奶量显著下降(从38.8 kg/d降至35.2 kg/d);而添加RPMet和RPLys后,产奶量恢复至36.9 kg/d,与饲喂高蛋白质饲粮时无显著差异,且组间乳蛋白率差异不显著。但也有研究显示,在低蛋白质饲粮中添加多种RPAA后,产奶量并未显著提升^[20-21]。本试验中,3组之间产奶量虽无显著差异,但从数值上看,AMP组与DMP+AA组较为接近,且均高于DMP组0.4 kg/d以上,这与多数前人研究结果的趋势一致。尽管本试验中DMP+AA组饲粮MP供应不足,但通过添加RPMet和RPLys,奶牛仍能维持正常的产奶量和乳蛋白产量,这可能是由于RPAA的补充弥补了MP不足对乳蛋白合成的限制。DMP组与AMP组的产奶量差异不显著,可能是因为DMP组饲粮的MP缺乏程度较轻(仅低于需要量8%)。此外,产奶量和乳蛋白产量还与泌乳阶段相关。Van den bossche等^[21]提出,泌乳中期奶牛的生理状态相对稳定,通常处于能量平衡状态,因此对饲粮CP水平变化的敏感性较低,这使得低蛋白质饲粮对其生产性能的影响不明显。乳糖是牛奶中的主要碳水化合物,在乳腺中,乳糖含量的升高会增加牛奶的渗透压,进而促进血液中的水分通过乳腺细胞进入牛奶,因此通常乳糖含量越高,产奶量也越高^[22]。本试验结果符合这一规律,从数值上看,乳糖率较高的组,其产奶量也相对较高。乳尿素氮含量与尿氮排出量存在强相关性,可用于预测奶牛的氮排泄量,是反映奶牛氮利用效率的重要指标。Arriola等^[23]在CP水平为15%的饲粮中补充1种或多种RPAA,结果显示,与CP水平为17%的饲粮组相比,奶牛产奶量无显著变化,但低蛋白质组的乳尿素氮含量显著降低,表明低蛋白质氨基酸平衡饲粮在一定程度上可提高氮利用率。本试验中,DMP组的乳尿素氮含量较AMP组极显著降低,与前人研究结果一致^[20-21]。这不仅是因为DMP组奶牛的氮摄入量相对较低,还表明其氮利用率较高,即乳腺中的氮更多地用于乳蛋白合成,而非转化为尿素氮。DMP+AA组的乳尿素氮含量较DMP组有所提高,表明补充RPMet和RPLys后,奶牛的氮利用率有所改善。

3.2 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛营养物质全肠道表观消化率的影响

全肠道表观消化率是反映奶牛对饲粮营养物质消化吸收状况的重要指标^[3]。研究表明,将饲粮CP水平从18%降至16.4%,不会对奶牛的营养物质消化率及饲喂效率产生影响^[24]。雒诚龙^[25]研究发现,将泌乳中后期奶牛饲粮CP水平从17%降至16%,不仅不影响饲喂效率,还能提高奶牛DM、CP及NDF表观消化率。然而,Kand等^[26]以蚕豆籽粒为主要蛋白质原料,设置高蛋白质组(CP水平为15.9%)和低蛋白质组(CP水平为12.8%)进行研究,结果显示低蛋白质组奶牛的采食量和NDF、CP表观消化率均显著降低。上述研究表明,适量降低饲粮CP水平对营养物质表观消化率无负面影响,但CP水平过低则会阻碍营养物质的消化。本试验中,AMP组与DMP组的DM、CP全肠道表观消化率无显著差异,与上述结论相符。添加RPAA后,DMP+AA组的DM全肠道表观消化率虽与DMP组无显著差异,但数值上有所提升,这与部分研究结果一致^[27-28]。本试验中,AMP组EE全肠道表观消化率极显著低于其余2组,可能是由于DMP组饲粮MP不足,机体上调脂肪分解酶活性,优先分解脂肪供能,从而提高了EE全肠道表观消化率^[29]。此外,AMP组较高的瘤胃液NH₃-N含量可能促使瘤胃微生物优先利用氮源合成自身蛋白,而非参与脂肪分解的代谢过程。

3.3 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛瘤胃发酵参数的影响

本试验结果表明,随着饲粮CP水平的下降,奶牛瘤胃液NH₃-N含量显著下降,这与前人研究结果相似^[28,30]。具体而言,AMP组饲粮提供了较高水平的RDP,其在瘤胃中被微生物分解后产生大量NH₃-N,导致瘤胃NH₃-N含量较高,达33.74 mg/dL;相比之下,DMP组由于饲粮RDP水平较低,瘤胃微生物对氮素的竞争性利用增强,减少了NH₃-N的生成及从瘤胃的溢出^[31],使其含量显著降低至28.21 mg/dL。此外,DMP+AA组通过补充RPMet和RPLys,优化了小肠氨基酸平衡,降低了宿主对瘤胃氮的依赖,从而减少了NH₃-N生成(降低至26.42 mg/dL),同时提高了氮利用率。这说明饲粮CP水平及氨基酸补充在调控瘤胃氮代谢中发挥着重要的作用。瘤胃液MCP含量取决于可消化营养物质的量及瘤胃微生物对这些营养物质的利用效率^[32]。研究显示,降低奶牛饲粮CP水平后,RDP供应不足会抑制瘤胃微生物生长,进而影响瘤胃发酵代谢,导致乙酸和丙酸含量下降^[33]。本试验结果表明,与AMP组相比,DMP组的瘤胃液MCP含量极显著降低,丙酸含量显著降低,乙酸含量在数值上也有所下降,与上述研究结果部分一致。AMP组因饲粮CP水平较高,瘤胃可消化底物充足;DMP+AA组则因DM的表观消化率较高,可能为微生物提供了更丰富的底物,因此这2组的瘤胃MCP含量相对较高。同时,DMP+AA组通过补充RPAA优化了小肠氨基酸平衡,减少了奶牛通过分解MCP获取必需氨基酸的需求,使更多MCP得以保留供自身利用,因此其MCP含量较DMP组极显著提高^[34]。本试验中,DMP和DMP+AA组的瘤胃液总挥发性脂肪酸含量极显著低于AMP组,提示DMP组可能因饲粮氮供应不足,降低了微生物可利用氮水平,从而阻碍了瘤胃中营养物质的消化与发酵。乙酸是乳脂合成的主要前体,而丙酸是肝脏糖异生的主要底物,经转化为葡萄糖后可支持产奶量并维持血糖稳态^[35],乙酸/丙酸升高会促进乳脂合成^[36]。本试验中,DMP组瘤胃液乙酸/丙酸较高,表明其更多能量被用于乳脂合成而非葡萄糖合成,这与该组乳脂率和乳脂产量数值较高的结果相符。

3.4 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛氮代谢的影响

一项荟萃分析研究表明,饲粮CP水平是影响奶牛氮利用率的最重要饲粮因素^[37]。Yan等^[38]也发现,氮摄入量是影响奶牛氮排泄量的最主要因素,且二者存在强正相关关系(决定系数=0.9)。本研究受试验条件限制,干物质采食量为群体平均采食量,由此计算的摄入氮数据无法进行统计分析,但从数值上看,DMP组的摄入氮低于AMP组,这与前人研究结果部分一致。大量研究显示,降低饲粮CP水平有助于减少奶牛的氮摄入量,而补充RPAA可提高奶牛氮利用率并减少氮排泄量^[3]。Lee等^[28,39]研究发现,在MP缺乏的饲粮中添加RPMet和RPLys后,与MP充足的饲粮相比,奶牛的生产性能和营养物质消化率无显著差异,但尿氮排泄量(减少49 g/d)、总氮排泄量及粪便可能释放的氨量均显著降低。赵若含等^[40]将泌乳前期奶牛饲粮CP水平从17.1%降至15.4%,同时添加9.4 g/d RPMet和25.7 g/d RPLys,结果显示,与对照组相比,试验组的粪氮和尿氮日排放量分别降低了10.2%和18.6%。本试验中,与AMP组相比,饲粮CP水平下降后,DMP和DMP+AA组的总排出氮、尿氮及尿氮占总排出氮比例均显著或极显著下降;且DMP+AA组的氮利用率较AMP组显著提高,与上述研究结果一致。结合乳尿素氮含量来看,AMP组因饲粮中MP和RDP充足,奶牛对尿素再循环利用的依赖降低,导致瘤胃NH₃-N溢出,肝脏尿素合成增加,更多氮以尿素形式排出体外,因此乳尿素氮、血浆尿素氮及尿尿素氮含量均较高。DMP组则因相反的机制减少了尿素氮的生成;而DMP+AA组由于补充了RPAA,促进了氨基酸平衡,一定程度上降低了奶牛对尿素循环利用的依赖,使其尿素合成水平介于其余2组之间。

3.5 低蛋白质饲粮补充RPMet和RPLys对泌乳奶牛血浆游离氨基酸含量的影响

小肠是氨基酸吸收的主要场所,氨基酸经肠道吸收进入血液后,通过血液循环被运输至乳腺、肝脏及其他组织。在泌乳奶牛体内,乳腺是氨基酸的主要消耗器官,乳腺细胞会摄取血液中的游离氨基酸用于乳蛋白合成^[41]。因此,血浆游离氨基酸含量在很大程度上能反映小肠可代谢氨基酸的数量及乳腺对氨基酸的摄取量,是评估奶牛氨基酸代谢状况的重要指标。有研究报道,当饲喂MP缺乏的饲粮时,奶牛血浆中部分必需氨基酸含量会降低。Giallongo等^[4]研究表明,MP缺乏组的血浆总必需氨基酸、精氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、苯丙氨酸和缬氨酸含量均低于MP充足组,蛋氨酸含量无显著变化;而添加RPMet、RPLys和过瘤胃组氨酸(RPHis)后,血浆组氨酸、赖氨酸含量显著提高,蛋氨酸含量在数值上有所提高。Lee等^[39]将奶牛饲粮MP供应量从需要量的100%降至87%,结果发现,血浆总必需氨基酸、赖氨酸、精氨酸和缬氨酸含量均显著降低,蛋氨酸含量无显著变化;在MP缺乏的饲粮中添加RPMet和RPLys后,血浆蛋氨酸含量显著提高,赖氨酸含量在数值上有所提高。Seleem等^[42]则发现,与高蛋白质饲粮组相比,在中等和较低蛋白质水平饲粮中添加RPMet和RPLys后,奶牛血浆中除脯氨酸外的其他氨基酸含量均无显著变化。本试验结果表明,与AMP组相比,DMP组的血浆总必需氨基酸含量虽仅在数值上减少,但蛋氨酸含量极显著下降,赖氨酸含量在数值上降低,这表明低蛋白质饲粮实际上会减少赖氨酸、蛋氨酸等必需氨基酸的供应量。血浆总必需氨基酸含量未出现显著降低,可能与饲粮设置的高、低CP水平(MP分别满足需要量的101%和92%)差距不够明显有关,也可能是试验过程中饲粮营养成分波动所致。DMP+AA组的血浆蛋氨酸含量较DMP组极显著提高,赖氨酸含量在数值上有所提高,说明RPAA的添加提高了这2种必需氨基酸的吸收利用量。其他氨基酸含量在各组间均无显著差异,与Seleem等^[42]的研究结果部分一致。

相关研究表明,补充的RPAA(如RPMet、RPLys)在小肠中可被直接吸收,从而显著提高血液中特定氨基酸的含量^[39,43],但也有部分研究仅观察到数值上的升高^[28,44-45]。不同研究中使用同种RPAA却出现结果差异,可能受以下因素影响:1)RPAA产品的实际生物有效性低于计算值^[46];2)奶牛生产阶段不同,例如围产期奶牛因能量负平衡和免疫激活,对限制性氨基酸的利用优先度可能从生产性能转向维持代谢稳态^[47];3)品种差异,荷斯坦奶牛因遗传选育形成的高泌乳特性,其小肠氨基酸吸收代谢反应与娟姗牛等品种存在差异,导致血浆氨基酸含量的响应不同^[48];4)饲养环境差异,有研究显示,热应激条件下奶牛的氨基酸需要量会增加,血液氨基酸含量下降,此时氨基酸可能更多地被用于转化为葡萄糖以满足能量需求^[49]。本试验中观察到补充RPAA后奶牛血浆氨基酸含量有响应,但生产性能无显著变化。这可能是由于试验开始前高估了奶牛饲粮的小肠可消化氨基酸供应量,导致尽管补充了RPMet和RPLys,氨基酸仍处于缺乏状态;同时,本试验仅额外补充了2种必需氨基酸,未考虑其他必需氨基酸的需要量,这一问题仍需进一步研究。

4 结论

基于新版NASEM(2021)模型对奶牛氨基酸营养需求进行评估,并据此在低蛋白质饲粮中补充RPMet和RPLys,可优化小肠氨基酸平衡,在数值上改善泌乳中期奶牛的产奶量,还能有效降低氮排泄量,尤其可减少尿氮的排放量及其占比,提高氮利用率。

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Publishing order | Descend order by publishing year | Descend order by cited within

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