Effects of Red Clover Isoflavones on Tolerance-Related Indices in Lactating Dairy Cows

ZHANG Shiqi; ZHANG Xiaoyin; XIONG Zhanbo; BU Ying; LI Kexin; ZHENG Nan; WANG Jiaqi; LIN Miao; ZHAO Guoqi; ZHAO Shengguo

doi:10.12418/CJAN2025.155

Chinese Journal of Animal Nutrition >

2025 , Vol. 37 >Issue 3: 1834 - 1843

DOI: https://doi.org/10.12418/CJAN2025.155

RESEARCH PAPER

Effects of Red Clover Isoflavones on Tolerance-Related Indices in Lactating Dairy Cows

ZHANG Shiqi ^,¹ ,
ZHANG Xiaoyin ¹ ,
XIONG Zhanbo ¹ ,
BU Ying ¹ ,
LI Kexin ¹ ,
ZHENG Nan ¹ ,
WANG Jiaqi ¹ ,
LIN Miao ² ,
ZHAO Guoqi ² ,
ZHAO Shengguo ^,¹^,^*

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¹ State Key Laboratory of Animal Nutrition and Feeding, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China
² College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China

*professor, E-mail: zhaoshengguo1984@163.com

Received date: 2024-07-26

Online published: 2025-03-13

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Abstract

This study aimed to investigate the effects of adding red clover isoflavones to diets on dairy cows’ performance and health, and to evaluate the tolerability of red clover isoflavones as feed additives. Twenty-four healthy Holstein lactating dairy cows with (2.13±1.12) parities, (168±23) d of lactation, and (30.73±2.70) kg of milk production were selected and divided into 3 groups of 8 cows each using a randomized complete-block design. The additive amounts of red clover isoflavones in diets of the 3 groups were 0 (control group), 0.8 and 8.0 g/kg DM, respectively. The experimental period was 56 days. The results showed that the addition of 0.8 and 8.0 g/kg DM of red clover isoflavones to the diets had no significant effect on body weight (P>0.05), but significantly reduced dry matter intake (week 4 and the whole trial period) compared with the control group (P<0.05); the addition of different amounts of red clover isoflavones did not have a significant effect on milk yield (P>0.05), but the milk lactose and non-fat milk solids contents were significantly higher in the 8.0 g/kg DM group than those in the control group (P<0.05). There were no significant differences in the white blood cell count, red blood cell count, hemoglobin concentration and erythrocyte pressure volume in the blood, and the contents of total protein, albumin, globulin, creatinine, glucose and total bilirubin and the activities of aspartate aminotransferase and alanine aminotransferase in the plasma of cows in the various groups (P>0.05). It is concluded that the addition of 0.8 and 8.0 g/kg DM of red clover isoflavones to diets has no negative effect on the performance and physiological health of lactating dairy cows. Therefore, lactating dairy cows can tolerate 8.0 g/kg DM of red clover isoflavones.

Key words： lactating dairy cows; red clover extract; red clover isoflavones; tolerance

Cite this article

ZHANG Shiqi , ZHANG Xiaoyin , XIONG Zhanbo , BU Ying , LI Kexin , ZHENG Nan , WANG Jiaqi , LIN Miao , ZHAO Guoqi , ZHAO Shengguo . Effects of Red Clover Isoflavones on Tolerance-Related Indices in Lactating Dairy Cows[J]. Chinese Journal of Animal Nutrition, 2025 , 37(3) : 1834 -1843 . DOI: 10.12418/CJAN2025.155

红三叶草,又名红车轴草,与苜蓿同属豆科,都为优良牧草。红三叶草富含异黄酮,主要为刺芒柄花素和鹰嘴豆芽素A^[1]。在北欧、北美等地区,红三叶草以干草和青贮的形式应用于反刍动物饲粮^[2]。研究表明,红三叶草及其活性成分在反刍动物养殖中具有积极的生物学功能。红三叶草青贮替代部分豆粕可提高瘤胃中必需氨基酸比例^[3],提高4%乳脂校正乳(FCM)产量^[4],提高饲料转化率和产奶量^[5],提高瘤胃微生物氮利用率^[6],提高羔羊瘤胃发酵效率^[7]。在反刍动物中,异黄酮可以提高生长性能,并可瘤胃调节微生物群落结构。鹰嘴豆芽素A可以提高奶山羊的产奶量、饲料转化率和产奶性能,并抑制高产氨菌活性,减少瘤胃发酵过程中的氮损失^[8]。在肉牛方面,鹰嘴豆芽素A可促进蛋白质消化和提高平均日增重,改善纤维分解菌对纤维的降解,避免亚急性瘤胃酸中毒,可作为传统抗生素替代品^[9]。综上可知,红三叶草及其活性成分对于反刍动物生产具有积极效果,开发其活性成分提取物作为饲料添加剂对于提升反刍动物生产性能具有积极意义。

红三叶草提取物(主要为红三叶草异黄酮)在体外发酵可显著降低尿素分解效率,提高瘤胃微生物蛋白含量,增加瘤胃蛋白质分解菌丰度,降低致病菌丰度,提高瘤胃微生物利用氨氮转化为微生物蛋白的能力^[10]。饲喂奶牛红三叶草提取物可提高产奶量、饲料转化率和氮利用效率等生产性能^[11],证明了红三叶草提取物作为饲料添加剂的有效性。异黄酮作为一种植物雌激素,可以调节动物或人类体内的雌激素平衡^[12⇓-14]。虽然奶牛的常规饲粮已包含如苜蓿干草等异黄酮来源,但作为饲料添加剂,饲粮外源性添加红三叶草异黄酮对奶牛健康状况的具体影响尚待明确。本试验旨在探究饲粮中添加红三叶草异黄酮对奶牛生产性能和机体健康的影响,对红三叶草异黄酮作为饲料添加剂的耐受性进行评价。

1 材料与方法

1.1 试验材料和试验许可

红三叶草提取物包括20%的红三叶草异黄酮(15.00%的刺芒柄花素+5.00%的鹰嘴豆芽素A)和80%的载体(载体由90%次粉和10%二氧化硅组成,载体营养成分含量:碳水化合物56.78%、粗灰分12.13%、粗脂肪1.77%、粗蛋白质9.12%、钠0.20%),以上数据均是基于干物质基础。试验在江苏省扬州高邮市扬州大学(高邮)试验牧场进行,所有程序均经中国农业科学院畜牧兽医研究所实验动物福利伦理委员会批准(编号:IAS 2022-107)。

1.2 试验设计

本试验选择24头胎次为(2.13±1.12)胎、泌乳天数为(168±23) d、产奶量为(30.73±2.70) kg的健康荷斯坦泌乳奶牛,采用随机区组设计,分为3组,每组8头。在基础饲粮中分别添加0、4、40 g/kg的红三叶草提取物,使得饲粮中红三叶草异黄酮添加剂量以饲粮干物质计分别为0(对照组)、0.8(0.59 g/kg刺芒柄花素+0.21 g/kg鹰嘴豆芽素A)和8.0 g/kg(6.02 g/kg刺芒柄花素+1.98 g/kg鹰嘴豆芽素A)。红三叶草异黄酮添加剂量的选择是根据《饲料和饲料添加剂畜禽靶动物耐受性评价试验指南》和有效性试验结果^[11]得出。有效性试验得出的最低推荐剂量为0.4 g/kg DM,根据奶牛的自身特性,选择最低推荐剂量的2倍作为有效剂量(0.8 g/kg DM),以有效剂量的10倍为高剂量(8.0 g/kg DM),进行奶牛饲喂试验,饲喂试验持续56 d。根据NRC(2001)奶牛营养需求配制基础饲粮,其组成及营养水平见表1。

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	30.00
苜蓿干草Alfalfa hay	13.50
全棉籽Whole cottonseed	5.65
玉米Corn	7.32
豆粕Soybean meal (46% CP)	10.90
熟麦皮Cooked wheat hulls	3.72
尿素Urea	0.54
压片玉米Flaked corn	19.40
膨化大豆Puffed soybean	3.01
脂肪粉Fat powder	0.82
碳酸氢钾KHCO₃	0.32
小苏打NaHCO₃	0.33
过瘤胃赖氨酸Rumen-protected lysine	0.17
过瘤胃蛋氨酸 Rumen-protected methionine	0.08
糖蜜Molasses	0.99
预混料Premix¹⁾	3.25
合计Total	100.00
营养水平Nutrient levels²⁾
粗蛋白质CP	18.30
淀粉Starch	24.10
中性洗涤纤维NDF	26.10
酸性洗涤纤维ADF	19.30
泌乳净能NE_L/(MJ/kg)	29.58

1)每千克预混料(干物质基础)提供 Each kg of premix (DM basis) provided the following:Ca 4 g,P 5.5 g,Na 2 g,Mg 4 g,VA 40 000 IU,VD 37 000 IU,VE 500 IU,Cu (as copper sulfate) 30 mg,Fe (as ferrous sulfate) 25 mg,Mn (as manganese sulfate) 140 mg,Zn (as zinc sulfate) 140 mg,Se (as sodium selenite) 0.80 mg。

2)除泌乳净能外的其他指标均为实测值。泌乳净能根据NRC(2001),使用整个试验期间奶牛的实际干物质采食量、产奶量、乳成分和体重估算得出。All other indexes except NE_L were measured values. NE_L was estimated based on NRC (2001) using actual dry matter intake, milk yield, milk composition, and body weight of the cows during the experiment.

1.3 饲喂管理与奶牛状况监察

奶牛自由采食、饮水,目标剩料量为10%。使用转台挤奶机(AutoRotor PerFormer Plus,GEM Inc.)挤奶。牛舍改装使得每组奶牛饲养在1个栅栏中。试验期内每天检查、记录奶牛的健康状况。

1.4 样品采集与指标测定

1.4.1 饲粮营养成分

收集新鲜的饲粮和剩料样品,65 ℃烘72 h后,再经105 ℃干燥4 h得到干物质含量。用粉碎机(Wiley Mill,托马斯科学公司,美国)粉碎干燥样品,并过1 mm筛。使用氮分析仪(CNS-2000型,LECO公司,美国)采用燃烧法[AOAC(2002)990.03]测定氮含量(N%),N%×6.25即为粗蛋白质含量。中性洗涤纤维和酸性洗涤纤维含量分别按照Van Soest等^[15]和Goering等^[16]的方法测定。淀粉含量按照Hendrix^[17]的方法(酶解法)测定。异黄酮含量参照Krenn等^[18]的方法,使用高效液相色谱仪[(Waters LC-2695,Waters XBridge C18色谱柱(250 mm×4.6 mm,5 μm)]测定,柱温为40 ℃,流速为1.0 mL/min,检测波长为270 nm,流动相包括乙腈(洗脱液A)和水/乙腈(75/25,v/v)混合物(洗脱液B)。

1.4.2 生产性能

在第0、4和8周的最后3 d记录采食量和计算剩料量,计算干物质采食量。在试验开始和第8周结束时用奶牛专用体尺估测各组奶牛体重。奶牛体重估计采用凯透罗氏(比利时)的估重公式,即:体重(kg)=[胸围(m)]²×体斜长(m)×90。

在试验第0、4和8周的最后3 d记录产奶量。分别于08:30、15:30和22:30,每头奶牛采集50 mL牛奶样品,并按4∶3∶3的比例混合均匀,加入装有溴-2-硝基丙烷-1,3-二醇(防腐剂)的冻存管中。4%FCM产量按照公式4%FCM产量=0.4×产奶量(kg/d)+15×乳脂肪产量(kg/d)计算得出。饲料转化效率按照公式饲料转化效率=4%FCM(kg/d)/平均干物质采食量(kg/d)计算得出^[19]。乳蛋白、乳脂肪、乳糖、乳总固形物和非脂乳固形物含量采用乳成分分析仪(MilkoScan FT120,FOSS公司,美国)进行测定。

1.4.3 血液指标

在试验第0、4和8周的最后1 d,从奶牛尾静脉采血2管(10 mL),为证明奶牛摄入红三叶草异黄酮后的代谢变化,采血时间选在晨饲后1 h,采血管分别含有肝素钠和乙二胺四乙酸(EDTA)。含EDTA的血液样本在4 ℃下保存,并在当天使用全自动血液分析仪(HemaVet,Drew Scientific,美国)进行分析,包括白细胞数量、红细胞数量、血红蛋白浓度、血细胞比容和血小板数量。含肝素钠的血液样本在3 000×g下离心15 min,上清液为血浆,吸取血浆并在-20 ℃冰箱中冷冻。血浆生化指标(总蛋白、白蛋白、球蛋白、尿素、肌酐、葡萄糖、总胆红素含量与谷草转氨酶、谷丙氨转氨酶、碱性磷酸酶活性)用Catalyst One全自动生化分析仪(Idexx Laboratories Inc.,美国)测定。

1.5 数据分析

试验数据经过Excel 2021进行初步处理后,使用SPSS 25.0的ANOVA程序进行单因素方差分析,并采用LSD法进行多重比较。结果数据用平均值和均值标准误(SEM)表示,以P<0.05为差异显著。

2 结果与分析

2.1 奶牛健康状况

整个试验期间,每天检查、记录奶牛的健康状况。各组奶牛在试验期间精神状态良好,采食和饮水等行为正常;8.0 g/kg DM组奶牛没有出现对红三叶草异黄酮不耐受的表现;各组奶牛没有发生流产、瘤胃酸中毒、跛行或临床乳房炎等疾病;试验期间没有奶牛死亡或被淘汰。

2.2 红三叶草异黄酮对泌乳奶牛生产性能的影响

由表2可知,添加不同剂量红三叶草异黄酮对泌乳奶牛的体重无显著影响(P>0.05)。在试验开始即第0周,各组的干物质采食量差异不显著(P>0.05);在第4周及整个试验全期(第0~8周),2个添加红三叶草异黄酮组奶牛的干物质采食量均显著降低(P<0.05);在第8周,8.0 g/kg DM组的干物质采食量显著低于对照组(P<0.05)。添加不同剂量的红三叶草异黄酮对泌乳奶牛各阶段的产奶量、4%FCM产量以及乳脂肪和乳蛋白含量无显著影响(P>0.05),8.0 g/kg DM组各阶段的乳糖、非脂乳固形物含量以及试验全期的乳总固形物含量显著高于对照组和0.8 g/kg DM组(P<0.05)。

表2 红三叶草异黄酮对泌乳奶牛生产性能的影响

Table 2 Effects of red clover isoflavones on performance of lactating dairy cows

项目 Items	红三叶草异黄酮添加剂量 Red clover isoflavones additive amount/(g/kg DM)			均值标准误 SEM	P值 P-value
项目 Items	0	0.8	8.0	均值标准误 SEM	P值 P-value
体重Body weight/kg
第0周Week 0	672.07	726.28	725.10	13.272	0.109
第8周Week 8	698.09	744.48	737.42	12.011	0.202
干物质采食量Dry matter intake/(kg/d)
第0周Week 0	17.97	18.35	19.08	0.213	0.065
第4周Week 4	21.84^a	21.00^b	20.37^b	0.196	0.003
第8周Week 8	23.61^a	23.20^ab	22.23^b	0.224	0.025
第0~8周Weeks 0 to 8	22.72^a	22.10^b	21.30^b	0.206	<0.001
产奶量Milk yield/(kg/d)
第0周Week 0	26.01	25.92	25.85	0.625	0.968
第4周Week 4	27.04	27.28	25.71	0.903	0.383
第8周Week 8	25.46	26.10	24.74	0.873	0.534
第0~8周Weeks 0 to 8	26.25	26.69	25.22	0.635	0.202
4%FCM产量4%FCM yield/(kg/d)
第0周Week 0	26.89	26.11	27.67	0.255	0.934
第4周Week 4	28.54	27.19	27.33	0.373	0.394
第8周Week 8	28.13	28.72	27.67	0.369	0.513
第0~8周Weeks 0 to 8	28.33	28.01	27.52	0.266	0.391
乳成分Milk composition/%
乳脂肪Milk fat
第0周Week 0	4.22	4.05	4.47	0.094	0.138
第4周Week 4	4.37	3.98	4.42	0.125	0.247
第8周Week 8	4.70	4.67	4.79	0.136	0.905
第0~8周Weeks 0 to 8	4.53	4.33	4.61	0.094	0.341
乳蛋白Milk protein
第0周Week 0	3.54	3.33	3.60	0.054	0.054
第4周Week 4	3.67	3.57	3.75	0.065	0.401
第8周Week 8	4.08	3.80	4.05	0.076	0.092
第0~8周Weeks 0 to 8	3.87	3.68	3.90	0.053	0.055
乳糖Milk lactose
第0周Week 0	4.52^b	4.59^b	4.79^a	0.055	0.048
第4周Week 4	4.46^b	4.53^b	4.82^a	0.053	0.004
第8周Week 8	4.46^b	4.64^b	4.84^a	0.053	0.002
第0~8周Weeks 0 to 8	4.46^b	4.58^b	4.83^a	0.048	<0.001
非脂乳固形物Nonfat milk solids
第0周Week 0	8.73^b	8.61^b	9.11^a	0.077	0.010
第4周Week 4	8.89^b	8.86^b	9.37^a	0.089	0.022
第8周Week 8	9.24^b	9.24^b	9.71^a	0.075	0.006
第0~8周Weeks 0 to 8	9.06^b	9.05^b	9.54^a	0.063	<0.001
乳总固形物Milk total solids
第0周Week 0	14.06	13.91	14.50	0.190	0.346
第4周Week 4	13.26	12.84	13.79	0.187	0.092
第8周Week 8	12.95^ab	12.66^b	13.59^a	0.149	0.021
第0~8周Weeks 0 to 8	13.10^b	12.75^b	13.69^a	0.120	0.002

	同行平均值肩标不同字母表示差异显著(P<0.05),相同字母或者无字母表示差异不显著(P>0.05)。第0~8周数据是第0、4、8周数据的最小二乘均值,表示整个试验期指标数值的变化。下表同。
	In the same row, the shoulders of mean with different letters indicated significant difference (P<0.05), while with the same letter or no letter indicated no significant difference (P>0.05). The data for weeks 0 to 8 are the least squares means of the data for weeks 0, 4 and 8, indicating the changes in the values of the indicators throughout the test period. The same as below.

2.3 红三叶草异黄酮对泌乳奶牛血液常规指标的影响

由表3可知,在第0、4周和8周以及试验全期,添加不同剂量的红三叶草异黄酮对泌乳奶牛白细胞数量、红细胞数量、血红蛋白浓度和红细胞压积均无显著影响(P>0.05)。在第0周,0.8 g/kg DM组和8.0 g/kg DM组的血小板数量显著低于对照组(P<0.05);在第4周,血小板数量各组间差异不显著(P>0.05);在第8周,8.0 g/kg DM组血小板数量与对照组相比无显著差异(P>0.05),而0.8 g/kg DM组则显著低于对照组(P<0.05);就试验全期而言,0.8 g/kg DM组和8.0 g/kg DM组血小板数量较对照组显著降低(P<0.05)。

表3 红三叶草异黄酮对泌乳奶牛血液常规指标的影响

Table 3 Effects of red clover isoflavones on blood routine indexes of lactating dairy cows

项目 Items	红三叶草异黄酮添加剂量 Red clover isoflavones additive amount/(g/kg DM)			均值标准误 SEM	P值 P-value
项目 Items	0	0.8	8.0	均值标准误 SEM	P值 P-value
白细胞数量WBC count/(×10⁹个/L)
第0周Week 0	10.84	12.84	8.99	0.972	0.314
第4周Week 4	11.46	11.90	10.96	0.763	0.899
第8周Week 8	10.68	11.90	11.01	0.784	0.829
第0~8周Weeks 0 to 8	10.99	12.21	10.32	0.846	0.682
红细胞数量RBC count/(×10¹²个/L)
第0周Week 0	5.87	6.07	5.45	0.256	0.642
第4周Week 4	6.12	6.16	6.34	0.089	0.568
第8周Week 8	6.91	6.14	7.34	0.203	0.051
第0~8周Weeks 0 to 8	6.30	6.12	6.38	0.184	0.423
血红蛋白浓度Hemoglobin concentration/(g/L)
第0周Week 0	94.13	96.63	91.50	4.224	0.899
第4周Week 4	100.13	98.50	103.50	1.752	0.513
第8周Week 8	114.13	110.00	119.13	4.114	0.697
第0~8周Weeks 0 to 8	102.80	101.71	104.71	3.365	0.701
血小板数量PLT count/(×10⁹个/L)
第0周Week 0	288.13^a	161.63^b	129.75^c	22.537	0.006
第4周Week 4	236.50	139.88	194.63	18.187	0.105
第8周Week 8	296.88^a	150.75^b	294.75^a	20.064	0.001
第0~8周Weeks 0 to 8	236.50^a	139.88^c	194.63^b	20.264	0.041
红细胞积压PCV/%
第0周Week 0	27.68	28.29	26.70	1.215	0.879
第4周Week 4	29.11	28.39	30.76	0.533	0.192
第8周Week 8	26.85	26.23	28.68	0.666	0.157
第0~8周Weeks 0 to 8	27.88	27.64	28.71	0.802	0.412

2.4 红三叶草异黄酮对泌乳奶牛血浆生化指标的影响

由表4可知,第4和8周时,添加不同剂量红三叶草异黄酮对泌乳奶牛血浆中总蛋白、白蛋白、球蛋白、肌酐、葡萄糖、总胆红素含量以及谷草转氨酶和谷丙转氨酶活性均无显著影响(P>0.05)。在第0和8周,添加不同剂量红三叶草异黄酮对泌乳奶牛血浆中尿素含量无显著影响(P>0.05),但是从第4周和试验全周期来看,8.0 g/kg DM组的血浆中尿素含量显著低于对照组和0.8 g/kg DM组(P<0.05)。

表4 红三叶草异黄酮对泌乳奶牛血浆生化指标的影响

Table 4 Effects of red clover isoflavones on plasma biochemical indexes of lactating dairy cows

项目 Items	红三叶草异黄酮添加剂量 Red clover isoflavones additive amount/(g/kg DM)			均值标准误 SEM	P值 P-value
项目 Items	0	0.8	8.0	均值标准误 SEM	P值 P-value
总蛋白Total protein/(g/L)
第0周Week 0	67.40	70.80	67.70	1.373	0.599
第4周Week 4	65.91	69.57	70.01	2.021	0.677
第8周Week 8	66.69	71.60	71.64	1.563	0.284
第0~8周Weeks 0 to 8	66.67	70.66	69.78	1.654	0.520
白蛋白Albumin/(g/L)
第0周Week 0	29.01	29.07	30.81	0.632	0.577
第4周Week 4	26.30	27.23	28.99	0.663	0.249
第8周Week 8	28.66	30.79	31.10	0.561	0.096
第0~8周Weeks 0 to 8	27.99	29.03	30.30	0.621	0.307
球蛋白Globulin/(g/L)
第0周Week 0	38.39	41.73	36.89	1.362	0.605
第4周Week 4	39.61	42.34	41.02	1.771	0.832
第8周Week 8	38.03	40.82	40.54	1.363	0.645
第0~8周Weeks 0 to 8	38.68	41.63	39.48	1.492	0.694
肌酐Creatinine/(μmol/L)
第0周Week 0	85.92	82.44	89.19	1.972	0.639
第4周Week 4	84.30	82.51	73.27	2.113	0.071
第8周Week 8	73.82	74.27	76.16	1.454	0.811
第0~8周Weeks 0 to 8	81.35	79.74	79.54	1.852	0.507
尿素Urea/(mmol/L)
第0周Week 0	5.23	5.37	5.80	0.192	0.370
第4周Week 4	5.28^a	5.51^a	4.02^b	0.251	0.020
第8周Week 8	6.09	5.25	5.34	0.203	0.137
第0~8周Weeks 0 to 8	5.53	5.38	5.05	0.213	0.176
葡萄糖Glucose/(mmol/L)
第0周Week 0	2.65	2.65	2.84	0.072	0.679
第4周Week 4	2.76	2.92	2.75	0.041	0.153
第8周Week 8	2.79	2.89	2.81	0.062	0.745
第0~8周Weeks 0 to 8	2.73	2.82	2.80	0.062	0.526
总胆红素Total bilirubin/(μmol/L)
第0周Week 0	1.95	1.73	1.77	0.072	0.480
第4周Week 4	1.94	1.62	2.06	0.133	0.380
第8周Week 8	1.90	1.90	1.80	0.101	0.772
第0~8周Weeks 0 to 8	1.93	1.75	1.88	0.103	0.544
谷草转氨酶Aspartate aminotransferase/(U/L)
第0周Week 0	67.40	70.80	67.70	1.382	0.599
第4周Week 4	65.91	69.57	70.01	2.022	0.677
第8周Week 8	66.69	71.60	71.64	1.562	0.284
第0~8周Weeks 0 to 8	66.67	70.66	69.78	1.653	0.520
谷丙转氨酶Alanine aminotransferase/(U/L)
第0周Week 0	29.01	29.07	30.81	0.632	0.577
第4周Week 4	26.3	27.23	28.99	0.663	0.249
第8周Week 8	28.66	30.79	31.10	0.562	0.096
第0~8周Weeks 0 to 8	27.99	29.03	30.30	0.622	0.307
碱性磷酸酶Alkaline phosphatase/(U/L)
第0周Week 0	38.39	41.73	36.89	1.362	0.605
第4周Week 4	39.61	42.34	41.02	1.762	0.832
第8周Week 8	38.03	40.82	40.54	1.363	0.645
第0~8周Weeks 0 to 8	38.68	41.63	39.48	1.491	0.694

3 讨论

本研究根据《饲料和饲料添加剂畜禽靶动物耐受性评价试验指南》的要求以及奶牛自身特性,选择0.8 g/kg DM作为红三叶草异黄酮的有效剂量,选用有效剂量的10倍(8.0 g/kg DM)为多倍剂量进行为期56 d的奶牛饲喂试验。由于奶牛屠宰成本较高,因此本试验未进行毒理学评价,只进行了生产性能监测、临床观察和血液学监测。结果发现,饲粮中添加0.8和8 g/kg DM的红三叶草异黄酮对奶牛的生产性能和生理健康无负面影响,可在不影响泌乳性能的情况下降低采食量,提高乳品质。

有关红三叶异黄酮对奶牛生产性能影响的研究较少。本研究结果表明,饲粮中添加红三叶草异黄酮降低了泌乳奶牛的干物质采食量。这一结果与红三叶草青贮降低了生长羔羊^[20]和泌乳奶牛^[21]的干物质采食量的研究结果一致。已有研究表明,血浆胰岛素水平随着饲粮红三叶草水平的增加而线性降低^[11]。因此,红三叶草异黄酮可能通过提高胰岛素敏感性^[22],从而降低分泌胰岛素的水平,进一步调节下丘脑中刺鼠关联蛋白(AgRP)/神经肽Y(NPY)的表达,从而调节干物质采食量^[23];此外,还可能是由于随着饲粮中红三叶草异黄酮剂量的增加,红三叶草异黄酮略苦的口感,尤其是大量添加时可能影响了适口性,以及提高了饲料转化效率,降低了奶牛采食需求。红三叶草异黄酮虽然降低了干物质采食量,但是对奶牛的产奶量无消极影响,并且添加8.0 g/kg DM红三叶草异黄酮还可增加乳糖和乳总固形物含量。在红三叶草异黄酮的有效性研究中表明,红三叶草异黄酮具有调节泌乳激素分泌和免疫功能的作用,使得奶牛采食量降低且不影响泌乳性能,并能提高饲料利用率,将添加剂量增加到8.0 g/kg DM时,仍然对生产性能无负面影响^[11]。

血液生化指标是反映机体健康状况的重要指标,其中总蛋白、白蛋白和球蛋白含量反映肝脏的合成功能,肌酐含量反映肾脏损伤情况,谷草转氨酶和谷丙转氨酶活性反映肝细胞通透性和代谢功能,是重要的肝脏功能指标,细胞渗漏和肝脏结构功能完整性丧失会引起血液中谷草转氨酶和谷丙转氨酶活性的增加^[24]。已有研究表明,奶山羊饲喂苜蓿和红三叶草异黄酮对血液中总蛋白、白蛋白、球蛋白、肌酐、葡萄糖及总胆红素含量无显著影响^[25],本试验结果与此一致。血液尿素含量与氮利用率成反比,已有研究表明红三叶草异黄酮鹰嘴豆芽素A提高了奶山羊的氮利用率,使得血液尿素含量降低。有文献报道,正常奶牛泌乳周期内血浆中尿素含量为3.6~14.0 mmol/L^[26],而本试验测得的泌乳奶牛血浆中尿素含量在此生理范围内。本试验中,添加红三叶草异黄酮的组泌乳奶牛血液中血小板数量在第0周时就有显著差异,可能是由于奶牛处于不同生理周期导致的,但是试验期奶牛的血小板数量在正常数量范围(100×10⁹~800×10⁹个/L)内^[27]。因此,红三叶草异黄酮未对泌乳奶牛血液生化指标产生负面影响。

相关研究显示,红三叶草中异黄酮含量为10.4~14.9 mg/g DM^[28]。Mustonen等^[29]研究表明,与黑麦草放牧条件相比,红三叶草放牧条件下奶牛的泌乳性能不会下降,同时还能降低尿氮排出;在这种情况下,奶牛平均红三叶草干物质采食量约为15.6 kg/d,红三叶草异黄酮摄入量为162.2~232.4 g/d。在本研究中,奶牛平均干物质采食量约为22 kg/d,0.8 g/kg DM组和8.0 g/kg DM组奶牛红三叶草异黄酮摄入量分别为17.6和176 g/d。由此可知,最高剂量(8.0 g/kg DM)组奶牛的红三叶草异黄酮摄入量与放牧条件下的红三叶草异黄酮摄入量相近。综上所述,饲粮中添加0.8~8.0 g/kg DM的红三叶草异黄酮对泌乳奶牛的生产性能和健康没有负面影响。

4 结论

饲粮中添加0.8和8.0 g/kg DM的红三叶草异黄酮对泌乳奶牛的生产性能和生理健康无负面影响。因此,泌乳奶牛可耐受8.0 g/kg DM的红三叶草异黄酮。

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