Effects of Dietary Cobalamin on Lactation Performance, Rumen Fermentation and Liver Gluconogenesis of Holstein Dairy Cows

HUANG Shi; YAN Guojun; CUI Hang; WANG Cong; LIU Qiang; GUO Gang; ZHANG Yawei

doi:10.12418/CJAN2024.092

2024 , Vol. 36 >Issue 2: 1020 - 1028

DOI: https://doi.org/10.12418/CJAN2024.092

RESEARCH PAPER

Effects of Dietary Cobalamin on Lactation Performance, Rumen Fermentation and Liver Gluconogenesis of Holstein Dairy Cows

HUANG Shi ^,¹ ,
YAN Guojun ² ,
CUI Hang ¹ ,
WANG Cong ^,¹^,^* ,
LIU Qiang ¹ ,
GUO Gang ¹ ,
ZHANG Yawei ¹

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¹ College of Animal Science, Shanxi Agricultural University, Taigu 030801, China
² Development Center of Rural Industry Integration, Department of Agriculture and Rural Affairs of Shanxi Province, Taiyuan 030001, China

^* professor, E-mail: wangdx0321@163.com

Received date: 2023-09-15

Online published: 2024-02-01

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Abstract

This experiment was conducted to investigate the effects of dietary cobalamin on lactation performance, rumen fermentation and liver gluconogenesis of Holstein dairy cows. Sixty Holstein dairy cows with similar body weight of (652.0±42.5) kg, parity of (2.04±0.39), lactation days of (104.7±31.2) and average milk yield of (33.6±18.7) kg/d were divided into 4 groups (15 cows per group) according to random block design, and the cobalamin was supplemented with 0 (control group), 6 (LCCA group), 12 (MCCA group) and 18 mg/d (HCCA group) in the form of coated cobalamin (CCA), respectively. The pre-trial period lasted for 20 days and the experimental period lasted for 60 days. The results showed as follows, with the increase of CCA supplemental level: 1) there was no significant difference in the body weight change of dairy cows (P>0.05), the dry matter intake was linearly decreased (P<0.05), and the yields of 4% fat correction milk and milk components (milk fat, milk protein and lactose) and feed efficiency were linearly increased (P<0.05); 2) the pH in rumen had no significant change (P>0.05), the total volatile fatty acid content and propionate molar percentage in rumen were linearly increased (P<0.05), and the acetate molar percentage and acetate to propionate ratio in rumen were linearly decreased (P<0.05); 3) the numbers of Ruminobacter amylophilus, Prevotella ruminicola, Butyrivibrio fibrisolvens, Ruminococcus albus, Ruminococcus flavefaciens, total fungi and total protozoa in rumen were increased linearly (P<0.05), while the numbers of Fibrobacter succinogenes and total bacteria in rumen were quadraticly changed (P<0.05); 4) the contents of glucose, total protein, albumin, insulin and cobalamin in serum were linearly increased (P<0.05), while the contents of urea nitrogen, triglyceride, non-esterification fatty acid and β-hydroxybutyrate in serum had no significant changes (P>0.05); 5) the activities of methylmalonyl-coenzyme A mutase, pyruvate carboxylase and phosphoenolpyruvate carboxykinase and the cobalamin content in liver were linearly increased (P<0.05), while the activities of glucose-6-phosphatase, fructose-1,6-bisphosphatase and glycogen content in liver had no significant changes (P>0.05). In conclusion, dietary CCA can improve lactation performance, rumen propionate content and liver gluconogenic key enzyme activities of dairy cows, and it is recommended that the supplemental level of cobalamin in the form of CCA is 12 mg/d in dairy cow diets.

Key words： dairy cows; cobalamin; lactation performance; rumen fermentation; gluconeogenesis

Cite this article

HUANG Shi , YAN Guojun , CUI Hang , WANG Cong , LIU Qiang , GUO Gang , ZHANG Yawei . Effects of Dietary Cobalamin on Lactation Performance, Rumen Fermentation and Liver Gluconogenesis of Holstein Dairy Cows[J]. Chinese Journal of Animal Nutrition, 2024 , 36(2) : 1020 -1028 . DOI: 10.12418/CJAN2024.092

钴胺素(cobalamin)即维生素B₁₂,其作为蛋氨酸合成酶和甲基丙二酰辅酶A变位酶(methylmalonyl-CoA mutase,MCM)的辅酶,参与一碳循环和糖异生代谢,是反刍动物瘤胃丙酸产生和肝脏糖异生的关键因子^[1];而促进瘤胃丙酸产生和肝脏糖异生是改善奶牛葡萄糖供应的有效途径。在瘤胃中,MCM催化琥珀酰辅酶A转化为甲基丙二酰辅酶A,这是丙酸形成的关键步骤^[1]。体外试验发现,饲粮添加钴胺素后,瘤胃原虫、纤维类分解菌和普雷沃氏菌数量增加^[2⇓-4],瘤胃总挥发性脂肪酸(total volatile fatty acid,TVFA)和丙酸含量提高^[4-5]。在肝脏中,MCM催化丙酸进入糖异生代谢的关键途径,在此过程中丙酰辅酶A异构化生成琥珀酰辅酶A^[6]。研究发现,羊钴胺素缺乏会导致肝脏MCM活性降低^[7],补充钴胺素后促进了羊肝脏中丙酸异生成葡萄糖的代谢^[8];奶牛补充钴胺素和叶酸后,肝脏MCM的表达上调^[9]。瘤胃微生物利用饲粮中的钴合成钴胺素,NRC(2001)^[10]推荐泌乳奶牛饲粮钴水平为0.11 mg/kg DM。但是,采食含钴0.11 mg/kg DM的饲粮,同时每周肌肉注射10 mg钴胺素,泌乳早期奶牛乳和乳成分产量提高^[11-12],血液钴胺素和总含硫氨基酸含量增加^[13]。试验结果显示,奶牛所需的钴胺素需要外源补充。饲粮中添加的钴胺素约62.9%在瘤胃中被微生物降解或破坏^[14],因此,反刍动物生产中应使用既能在瘤胃中释放钴胺素,又能使添加的钴胺素有效到达小肠的包被钴胺素(coated cobalamin,CCA)添加剂。目前,关于CCA对奶牛泌乳性能、瘤胃发酵和糖异生代谢影响的研究还未见报道。因此,本试验通过在饲粮中添加不同水平的CCA,探究其对奶牛泌乳性能、瘤胃发酵、瘤胃微生物数量、血液指标和肝脏糖异生的影响,以明确CCA对奶牛泌乳性能的调控特点及其可能的机制。

1 材料与方法

1.1 试验设计

本试验于2022年5—8月在山西省忻州银山湖奶牛养殖有限公司进行。试验选取体重[(652.0±42.5) kg]、胎次[(2.04±0.39)胎]、泌乳天数[(104.7±31.2) d]和平均产奶量[(33.6±18.7) kg/d]相近的荷斯坦奶牛60头,依据随机区组设计分为4组,每组15头牛。4个组分别以CCA的形式补充0(对照组)、6(LCCA组)、12(MCCA组)和18 mg/d(HCCA组)的钴胺素。试验用CCA由山西某科技有限公司生产,其中钴胺素含量为2%,瘤胃和小肠钴胺素释放率采用尼龙袋法,通过装有瘤胃和十二指肠瘘管的奶牛测定,分别为24.8%和70.5%。钴胺素的添加水平依据NRC(2001)^[10]中4%乳脂校正乳(FCM)产量为35 kg和体重为650 kg的奶牛的钴胺素推荐量0.6 mg/d,以及奶牛钴胺素的肠道吸收率10%~15%^[14]确定。饲养试验包括20 d预试期和60 d正试期。

1.2 试验饲粮和饲养管理

试验牛基础饲粮按照NRC(2001)^[10]配制,其组成及营养水平见表1;饲粮中钴含量采用原子吸收仪测定,各养分含量依据AOAC(2006)^[15]方法测定。试验牛于同一圈舍饲养,上颈枷饲喂,每日饲喂2次,挤奶3次,自由采食和饮水。早晨饲喂前,将CCA与约500 g全混合日粮(TMR)混匀,待牛全部采食后再饲喂剩余饲粮。

表1 基础饲粮组成及营养水平(干物质基础)

Table 1 Composition and nutrient levels of the basal diet (DM basis)%

项目 Items	含量 Content
原料 Ingredients
玉米青贮 Corn silage	26.00
苜蓿干草 Alfalfa hay	11.60
燕麦草 Oat hay	12.50
玉米 Corn	25.00
麸皮 Wheat bran	6.00
豆粕 Soybean meal	9.50
菜籽粕 Rapeseed meal	2.60
棉籽粕 Cottonseed meal	5.00
碳酸氢钙 Ca(HCO₃)₂	0.50
氯化钠 NaCl	0.50
磷酸氢钙 CaHPO₄	0.30
预混料 Premix¹⁾	0.50
合计 Total	100.00
营养水平 Nutrient levels²⁾
有机物 OM	94.60
粗蛋白质 CP	16.80
粗脂肪 EE	3.43
中性洗涤纤维 NDF	31.33
酸性洗涤纤维 ADF	19.24
钙 Ca	0.73
磷 P	0.45
钴 Co/(mg/kg)	0.21
泌乳净能 NE_L/(MJ/kg)	6.64

1)每千克预混料含有 One kg premix contained the following:Fe 20 000 mg,Cu 1 600 mg,Mn 8 000 mg,Zn 7 500 mg,I 120 mg,Se 60 mg,Co 20 mg,VA 820 000 IU,VD 300 000 IU,VE 10 000 IU。

2)泌乳净能依据NRC(2001)^[10]计算,计算公式为:泌乳净能(MJ/kg)=[0.024 5×饲粮可消化总养分(%)-0.12]×4.184,其余为实测值。NE_L was calculated according to NRC (2001)^[10], and the calculation formula was NE_L (MJ/kg)=[0.024 5×TDN(%)-0.12]×4.184, while the others were measured values.

1.3 测定指标及方法

1.3.1 泌乳性能的测定

正试期开始和结束时,每次持续2 d,早晨挤奶后,称每头牛空腹体重,计算试验期间体重变化(body weight change,BWC)。每天记录每头牛的喂料量与剩料量,每隔10 d,采集TMR和剩料样1次,依据AOAC(2006)^[15]方法测定干物质(DM)含量,计算每头牛每天的干物质采食量(DMI)。记录每头牛每天产奶量;每隔10 d,采集每头牛奶样,依据3次挤奶的产奶量,以牛为单位,将当天奶样混合,保存于装有2-溴-2-硝基-1,3-丙二醇的100 mL奶样瓶中,用红外乳品分析仪(MSF120,丹麦)测定脂肪、蛋白质和乳糖含量。

1.3.2 瘤胃液样品的采集与测定

正试期第57和58天,早晨饲喂后3~4 h,用胃管采样器采集每头牛瘤胃液样品约100 mL(为避免唾液污染,先采集的约200 mL瘤胃液弃掉),立即测定瘤胃液pH,然后将瘤胃液用4层医用纱布过滤,收集滤液。滤液一份保存于-20 ℃,采用内标法并利用气相色谱仪(GC9720,中国)测定各挥发性脂肪酸(volatile fatty acid,VFA)含量^[15];另一份保存于-80 ℃,用荧光定量PCR法测定各微生物数量,微生物引物序列由某公司合成,PCR反应体系依据试剂盒[宝生物工程(大连)有限公司]说明书设定。

1.3.3 血液样品的采集与测定

正试期第29和59天,挤奶后,晨饲前,采集每头牛尾静脉血液样品15~20 mL,在2 000×g和4 ℃条件下离心15 min制备血清,于-20 ℃保存。依据试剂盒(上海笃玛生物科技有限公司)说明书,用血液分析仪(Konelab PRIME 60,芬兰)测定血清中葡萄糖、总蛋白、白蛋白、尿素氮、甘油三酯、非酯化脂肪酸(NEFA)、β-羟丁酸(BHB)、胰岛素(INS)和钴胺素含量。

1.3.4 肝脏样品的采集与测定

正试期第60天,晨饲后,每组随机选取5头奶牛,在牛体右侧第10与11肋骨之间,用活体组织穿刺针采集肝脏样品0.5~1.0 g^[16]。组织样分2份立即存放于液氮中,然后转入-80 ℃冰箱。肝脏组织用全自动样品快速研磨仪匀浆后,离心,收集上清液用于酶活性测定。样品中MCM、丙酮酸羧化酶(PC)、葡萄糖-6-磷酸酶(G6P)、磷酸烯醇式丙酮酸激酶(PEPCK)和果糖-1,6-二磷酸酶(FBP)活性依据酶联免疫试剂盒说明书测定,酶活性表示为每克湿组织每分钟转化生成的底物或产物的量(μmol)。肝脏组织匀浆后,按试剂盒说明书测定每克湿组织中糖原和钴胺素的含量。肝脏样品测定试剂盒均购于上海笃玛生物科技有限公司。

1.4 数据统计分析

采用SPSS 22.0统计软件中的ANOVA过程对试验数据进行单因素方差分析,并采用Duncan氏法进行组间多重比较,试验结果以平均值和均值标准误(SEM)表示,P<0.05为差异显著。

2 结果

2.1 钴胺素对奶牛泌乳性能的影响

由表2可知,随着CCA添加水平的提高,奶牛BWC、乳蛋白质含量和乳糖含量无显著变化(P>0.05),DMI呈线性降低(P<0.05),实际产奶量呈二次变化(P<0.05),4%FCM产量、乳脂肪产量、乳蛋白质含量、乳糖产量、乳脂肪含量和饲料效率(feed efficiency,FE)呈线性提高(P<0.05)。其中,HCCA组奶牛DMI显著低于对照组、LCCA组和MCCA组(P<0.05);LCCA组和MCCA组实际产奶量显著高于对照组和HCCA组(P<0.05);MCCA组和HCCA组4%FCM产量和乳脂肪产量显著高于对照组(P<0.05);MCCA组乳蛋白质产量显著高于对照组(P<0.05);LCCA组和MCCA组乳糖产量显著高于对照组(P<0.05);HCCA组乳脂肪含量显著高于对照组(P<0.05);3个CCA添加组FE显著高于对照组(P<0.05)。

表2 钴胺素对奶牛泌乳性能的影响

Table 2 Effects of cobalamin on lactation performance of dairy cows

项目 Items	组别 Groups				SEM	P值 P-value
项目 Items	对照 Control	LCCA	MCCA	HCCA	SEM	处理 Treatment	线性 Linear	二次 Quadratic
初始体重 Initial BW/kg	650.3	653.7	657.4	649.1	42.53	0.213	0.710	0.634
体重变化 BWC/(kg/d)	0.26	0.29	0.37	0.36	0.024	0.117	0.067	0.549
干物质采食量 DMI/(kg/d)	21.47^a	21.60^a	21.28^a	20.52^b	0.115	0.003	0.001	0.073
产奶量 Milk yield/(kg/d)
实际产奶量 Actual milk yield	33.94^b	35.71^a	35.97^a	34.05^b	0.241	0.001	0.756	0.001
4%乳脂校正乳产量 4% FCM yield	28.44^b	30.40^ab	32.17^a	31.00^a	0.445	0.024	0.016	0.070
乳脂肪产量 Milk fat yield	0.99^b	1.07^ab	1.18^a	1.15^a	0.027	0.045	0.013	0.312
乳蛋白质产量 Milk protein yield	1.07^b	1.14^ab	1.17^a	1.13^ab	0.012	0.038	0.032	0.055
乳糖产量 Lactose yield	1.91^b	2.05^a	2.04^a	1.95^ab	0.018	0.015	0.002	0.056
乳成分 Milk component/%
乳脂肪 Milk fat	2.92^b	3.00^ab	3.29^ab	3.42^a	0.079	0.047	0.011	0.870
乳蛋白质 Milk protein	3.17	3.19	3.27	3.34	0.030	0.203	0.073	0.687
乳糖 Lactose	5.65	5.75	5.68	5.72	0.029	0.639	0.615	0.608
饲料效率 Feed efficiency	1.32^b	1.41^a	1.51^a	1.51^a	0.005	0.001	0.001	0.421

	同行数据肩标不同字母表示差异显著(P<0.05)。下表同。
	In the same row, values with different letter superscripts mean significant difference (P<0.05). The same as below.

2.2 钴胺素对奶牛瘤胃发酵参数和微生物数量的影响

由表3可知,随着CCA添加水平的提高,奶牛瘤胃pH以及丁酸、戊酸、异丁酸和异戊酸摩尔百分比无显著变化(P>0.05),瘤胃TVFA含量和丙酸摩尔百分比呈线性提高(P<0.05),瘤胃乙酸摩尔百分比和乙酸/丙酸值呈线性降低(P<0.05)。其中,MCCA组瘤胃TVFA含量最高,显著高于其他各组(P<0.05);其次为LCCA组和HCCA组,显著高于对照组(P<0.05)。MCCA组瘤胃乙酸摩尔百分比显著低于对照组(P<0.05)。LCCA组、MCCA组和HCCA组瘤胃丙酸摩尔百分比显著高于对照组(P<0.05)。MCCA组和HCCA组瘤胃乙酸/丙酸值显著低于对照组(P<0.05)。

表3 钴胺素对奶牛瘤胃发酵参数的影响

Table 3 Effects of cobalamin on rumen fermentation parameters of dairy cows

项目 Items	组别 Groups				SEM	P值 P-value
项目 Items	对照 Control	LCCA	MCCA	HCCA	SEM	处理 Treatment	线性 Linear	二次 Quadratic
pH	6.76	6.70	6.58	6.68	0.052	0.082	0.075	0.069
总挥发性脂肪酸 TVFA/(mmol/L)	124.80^c	127.80^b	135.40^a	129.70^b	1.180	0.001	0.006	0.087
乙酸 Acetate/%	55.04^a	54.43^ab	53.76^b	54.32^ab	0.435	0.025	0.036	0.064
丙酸 Propionate/%	24.32^b	25.47^a	26.15^a	26.45^a	0.312	0.021	0.013	0.063
丁酸 Butyrate/%	16.23	15.82	15.79	16.43	0.271	0.157	0.152	0.061
戊酸 Valerate/%	1.97	1.86	1.93	2.13	0.064	0.251	0.171	0.114
异丁酸 Isobutyrate/%	0.48	0.44	0.40	0.59	0.029	0.177	0.292	0.059
异戊酸 Isovalerate/%	1.94	1.95	1.96	2.05	0.065	0.382	0.147	0.314
乙酸/丙酸 Acetate/propionate	2.26^a	2.14^ab	2.06^b	2.05^b	0.046	0.023	0.014	0.052

由表4可知,随着CCA添加水平的提高,奶牛嗜淀粉瘤胃杆菌、栖瘤胃普雷沃氏菌、溶纤维丁酸弧菌、白色瘤胃球菌、黄色瘤胃球菌、总真菌和总原虫数量呈线性增加(P<0.05),瘤胃产琥珀酸丝状杆菌和总细菌数量呈二次变化(P<0.05)。其中,MCCA组瘤胃嗜淀粉瘤胃杆菌、栖瘤胃普雷沃氏菌、溶纤维丁酸弧菌和产琥珀酸丝状杆菌数量最多,显著高于其他组(P<0.05);LCCA组和HCCA组次之,显著高于对照组(P<0.05)。MCCA组瘤胃白色瘤胃球菌数量最多,显著高于其他组(P<0.05);HCCA组次之,显著高于LCCA组和对照组(P<0.05)。MCCA组和HCCA组瘤胃黄色瘤胃球菌数量较高,显著高于其他组(P<0.05);LCCA组次之,显著高于对照组(P<0.05)。MCCA组瘤胃总细菌数量显著高于HCCA组和对照组(P<0.05)。HCCA组瘤胃总真菌数量最高,LCCA组和MCCA组次之,对照组最低,但各组间无显著差异(P>0.05)。LCCA组、MCCA组和HCCA组瘤胃总原虫数量显著高于对照组(P<0.05)。

表4 钴胺素对奶牛瘤胃微生物数量的影响

Table 4 Effects of cobalamin on rumen microbial count of dairy cows

项目 Items	组别 Groups				SEM	P值 P-value
项目 Items	对照 Control	LCCA	MCCA	HCCA	SEM	处理 Treatment	线性 Linear	二次 Quadratic
嗜淀粉瘤胃杆菌 Ruminobacter amylophilus/ (×10⁸ copies/mL)	5.77^c	6.48^b	7.69^a	6.90^b	0.117	0.014	0.018	0.220
栖瘤胃普雷沃氏菌 Prevotella ruminicola/ (×10⁹ copies/mL)	4.22^c	4.77^b	5.30^a	4.71^b	0.163	0.047	0.016	0.068
溶纤维丁酸弧菌 Butyrivibrio fibrisolvens/ (×10⁸ copies/mL)	3.59^c	3.93^b	4.74^a	4.13^b	0.134	0.001	0.006	0.051
产琥珀酸丝状杆菌 Fibrobacter succinogenes/ (×10⁹ copies/mL)	5.40^c	6.78^b	7.76^a	6.29^b	0.317	0.023	0.140	0.013
白色瘤胃球菌 Ruminococcus albus/ (×10⁸ copies/mL)	3.25^c	3.45^c	5.11^a	4.06^b	0.305	0.035	0.026	0.259
黄色瘤胃球菌 Ruminococcus flavefaciens/ (×10⁹ copies/mL)	1.19^c	1.75^b	2.24^a	2.15^a	0.115	0.001	0.001	0.234
总细菌 Total bacteria/(×10¹¹ copies/mL)	4.79^b	5.65^ab	6.23^a	5.07^b	0.202	0.022	0.355	0.007
总真菌 Total fungi/(×10⁷ copies/mL)	4.61	5.15	5.54	6.35	0.240	0.097	0.016	0.747
总原虫 Total protozoa/(×10⁵ copies/mL)	1.37^b	1.76^a	1.84^a	2.04^a	0.121	0.039	0.015	0.719

2.3 钴胺素对奶牛血液指标的影响

由表5可知,随着CCA添加水平的提高,奶牛血清葡萄糖、总蛋白、白蛋白、INS和钴胺素含量呈线性提高(P<0.05),血清尿素氮、甘油三酯、NEFA和BHB含量无显著变化(P>0.05)。其中,LCCA组、MCCA组和HCCA组血清葡萄糖、总蛋白和白蛋白含量显著高于对照组(P<0.05);MCCA组和HCCA组血清INS和钴胺素含量显著高于对照组(P<0.05)。

表5 钴胺素对奶牛血液指标的影响

Table 5 Effects of cobalamin on blood indices of dairy cows

项目 Items	组别 Groups				SEM	P值 P-value
项目 Items	对照 Control	LCCA	MCCA	HCCA	SEM	处理 Treatment	线性 Linear	二次 Quadratic
葡萄糖 Glucose/(mmol/L)	3.41^b	3.96^a	4.14^a	4.07^a	0.082	0.019	0.003	0.463
总蛋白 Total protein/(g/L)	81.83^b	95.83^a	98.23^a	93.57^a	2.413	0.047	0.031	0.232
白蛋白 Albumin/(g/L)	46.56^b	53.06^a	55.70^a	52.30^a	1.630	0.029	0.017	0.148
尿素氮 Urea nitrogen/(mmol/L)	7.40	7.26	7.06	6.62	0.207	0.591	0.217	0.732
甘油三酯 Triglyceride/(mmol/L)	0.19	0.26	0.27	0.21	0.046	0.248	0.553	0.058
非酯化脂肪酸 NEFA/(μmol/L)	284.30	271.00	259.00	267.00	21.620	0.091	0.281	0.169
β-羟丁酸 BHB/(μmol/L)	786.60	765.00	777.30	783.00	17.950	0.425	0.740	0.516
胰岛素 INS/(mU/L)	8.85^b	11.58^ab	15.08^a	14.76^a	1.183	0.043	0.019	0.329
钴胺素 Cobalamin/(pmol/L)	304.40^b	321.10^ab	346.70^a	364.80^a	16.180	0.031	0.045	0.992

2.4 钴胺素对奶牛肝脏糖异生的影响

由表6可知,随着CCA添加水平的提高,奶牛肝脏MCM、PC和PEPCK活性以及钴胺素含量呈线性提高(P<0.05),肝脏G6PC和FBP活性以及糖原含量无显著变化(P>0.05)。其中,MCCA组和HCCA组肝脏MCM活性显著高于对照组(P<0.05);LCCA组、MCCA组和HCCA组肝脏钴胺素含量以及PC和PEPCK活性显著高于对照组(P<0.05)。

表6 钴胺素对奶牛肝脏糖异生的影响

Table 6 Effects of cobalamin on liver gluconeogenesis of dairy cows

项目 Items	组别 Groups				SEM	P值 P-value
项目 Items	对照 Control	LCCA	MCCA	HCCA	SEM	处理 Treatment	线性 Linear	二次 Quadratic
甲基丙二酰辅酶A变位酶 MCM/(U/g)	9.47^b	13.11^ab	16.18^a	15.23^a	1.836	0.029	0.037	0.178
丙酮酸羧化酶 PC/(U/g)	7.57^b	13.23^a	17.88^a	16.37^a	1.418	0.011	0.041	0.191
葡萄糖-6-磷酸酶 G6PC/(U/g)	6.18	6.52	6.48	6.31	0.168	0.927	0.862	0.746
磷酸烯醇式丙酮酸激酶 PEPCK/(U/g)	9.13^b	10.45^a	10.77^a	10.51^a	0.307	0.045	0.022	0.433
果糖-1,6-二磷酸酶 FBP/(U/g)	5.69	5.83	5.69	5.47	0.337	0.748	0.652	0.877
糖原 Glycogen/(mg/g)	19.43	18.69	21.01	20.87	1.643	0.186	0.438	0.321
钴胺素 Cobalamin/(μg/g)	1.07^b	1.21^a	1.16^a	1.30^a	0.032	0.004	0.012	0.317

3 讨论

3.1 钴胺素对奶牛泌乳性能的影响

本试验中,以CCA形式补充6或12 mg/d钴胺素,奶牛DMI无显著变化。其他研究同样发现,每周肌肉注射10 mg钴胺素对泌乳早期奶牛DMI无显著影响^[17-18]。不过,以CCA形式补充18 mg/d钴胺素,奶牛DMI显著降低,表明该水平对泌乳中期奶牛来讲可能太高。然而,Graulet等^[13]研究发现,饲粮中添加500 mg/d的钴胺素,奶牛DMI无显著变化。试验结果的差异,可能与钴胺素的补充方式不同有关。奶牛饲粮中直接添加的钴胺素,约62.9%在瘤胃中被微生物利用或破坏,使得到达小肠被吸收的钴胺素量减少^[14]。本试验所用的CCA添加剂采用pH敏感材料作为壁材,CCA中的钴胺素在瘤胃和小肠中的释放率分别为24.8%和70.5%。

不过,随着CCA添加水平的提高,虽然奶牛DMI呈线性降低,但是4% FCM和乳成分产量呈线性提高。试验结果表明,补充CCA改善了饲粮养分利用率,表现为FE提高。此外,试验期间奶牛BWC以及血清NEFA和BHB含量无显著变化,表明乳和乳成分产量的提高没有引起体脂动员增多。类似的研究报道,钴胺素和叶酸共同补充能缓减泌乳早期奶牛的能量负平衡^[19],奶牛产奶量的增加与能量代谢的改善有关^[11-12]。4%FCM和乳脂肪产量提高,与瘤胃乙酸含量的提高有关。对照组、LCCA组、MCCA组和HCCA组瘤胃乙酸含量分别为68.69、69.56、72.79和70.45 mmol/L。乙酸是乳脂肪酸合成的主要前体,增加奶牛乙酸供应能刺激乳脂肪酸合成,进而提高乳脂肪产量^[20]。乳蛋白质产量提高,与血清总蛋白和白蛋白含量提高的结果一致,表明补充CCA改善了饲粮蛋白质的利用率。一碳代谢中,钴胺素依赖的蛋氨酸合成酶催化同型半胱氨酸转化为蛋氨酸^[1]。蛋氨酸是奶牛乳蛋白质合成的必需氨基酸^[10]。Preynat等^[21]研究发现,补充叶酸的奶牛同时提供钴胺素,乳蛋白质产量提高。乳糖产量提高,与血清葡萄糖含量提高有关。葡萄糖是乳糖合成的前体物质,是乳腺组织的主要能量来源^[22]。对泌乳早期奶牛的研究同样发现,每周肌肉注射10 mg钴胺素,乳和乳成分产量增加^[11-12]。

3.2 钴胺素对奶牛瘤胃发酵参数和微生物数量的影响

奶牛饲粮中补充CCA,瘤胃TVFA含量提高,表明养分在瘤胃的降解增多,这与总细菌、总真菌和总原虫数量的增加有关。其中,瘤胃乙酸含量的增加,与真菌、原虫和主要纤维降解菌(溶纤维丁酸弧菌、产琥珀酸丝状杆菌、白色瘤胃球菌和黄色瘤胃球菌)数量的增加有关。在瘤胃中,真菌、原虫和纤维降解菌分泌纤维类分解酶,将降解结构性碳水化合物为乙酸^[23]。瘤胃丙酸含量提高则与嗜淀粉瘤胃杆菌、栖瘤胃普雷沃氏菌和溶纤维丁酸弧菌数量增加有关,这3种菌是瘤胃中的主要淀粉分解菌,通过分泌淀粉酶将饲粮中的非结构性碳水化合物降解为丙酸^[24]。瘤胃乙酸/丙酸值降低,表明瘤胃发酵类型转化为产生更多丙酸的丙酸型发酵。在瘤胃丙酸产生过程中,MCM催化琥珀酰辅酶A转化为甲基丙二酰辅酶A^[1]。体外试验同样发现,添加钴胺素促进了瘤胃原虫、纤维类分解菌和普雷沃氏菌的生长^[2⇓-4],提高了TVFA和丙酸含量^[4-5]。

3.3 钴胺素对奶牛血液指标和肝脏糖异生的影响

本试验中,随着CCA添加水平的提高,奶牛血清葡萄糖含量提高,这与肝脏糖异生关键酶MCM、PC和PEPCK活性提高的结果一致,表明补充CCA改善了奶牛肝脏糖异生代谢,且这与钴胺素在瘤胃丙酸产生和肝脏糖异生代谢中的营养生理功能有关。丙酸是糖异生的主要前体物质,钴胺素依赖的MCM催化丙酰辅酶A转化为琥珀酰辅酶A是丙酸进入糖异生代谢的关键步骤^[6]。已有报道,增加丙酸供应,肉牛肝脏葡萄糖产量增加^[25];补充钴胺素,促进了羊肝脏中丙酸异生成葡萄糖^[8]。类似的研究发现,奶牛同时补充钴胺素和叶酸,血液葡萄糖含量增加,整体葡萄糖流量有提高的趋势^[13,21];羊缺乏钴胺素,肝脏MCM活性降低^[7];奶牛同时补充钴胺素和叶酸,肝脏MCM表达上调^[9]。然而,Graulet等^[13]研究发现,泌乳奶牛补充500 mg/d的钴胺素,血清葡萄糖含量和肝脏MCM活性无显著变化。试验结果的不同可能与钴胺素的添加模式不同有关。补充CCA后,奶牛血清INS含量提高,这与血清葡萄糖含量提高有关,与肝脏PC和PEPCK活性提高的结果不一致。然而,已有研究证实,牛的丙酸糖异生代谢不受INS调控^[26]。然而,要明确CCA对奶牛葡萄糖供应的调控及机制还需进一步研究。本研究中,奶牛血清和肝脏钴胺素含量提高,表明补充CCA改善了泌乳奶牛钴胺素状态,而且6、12和18 mg/d的钴胺素补充水平对改善奶牛钴胺素状态无显著差异。考虑到18 mg/d钴胺素添加组奶牛DMI显著降低,12 mg/d钴胺素添加组瘤胃TVFA含量显著高于6 mg/d钴胺素添加组,推荐奶牛饲粮中以CCA形式补充钴胺素的添加水平为12 mg/d。

4 结论

饲粮中添加CCA使得奶牛乳和乳成分产量提高,这与瘤胃丙酸含量和肝脏糖异生关键酶活性提高有关;本试验条件下,推荐以CCA形式补充钴胺素的添加水平为12 mg/d。

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