亚急性瘤胃酸中毒对绵羊瘤胃异常代谢产物、瘤胃菌群和血液相关指标的影响

引用本文

韩郭皓, 高晓莎, 段晋伟, 张慧琴, 郑岩, 马林峰, 何金鑫, 霍乃蕊, 裴彩霞, 古少鹏. 亚急性瘤胃酸中毒对绵羊瘤胃异常代谢产物、瘤胃菌群和血液相关指标的影响[J]. 动物营养学报, 2021, 33(4): 2158-2167.

HAN Guohao, GAO Xiaosha, DUAN Jinwei, ZHANG Huiqin, ZHENG Yan, MA Linfeng, HE Jinxing, HUO Nairui, PEI Caixia, GU Shaopeng. Effects of Subacute Ruminal Acidosis on Rumen Abnormal Metabolites, Rumen Bacterial Flora and Blood Related Indexes of Sheep[J]. Chinese Journal of Animal Nutrition, 2021, 33(4): 2158-2167.

亚急性瘤胃酸中毒对绵羊瘤胃异常代谢产物、瘤胃菌群和血液相关指标的影响

韩郭皓¹ , 高晓莎¹ , 段晋伟¹ , 张慧琴¹ , 郑岩¹ , 马林峰¹ , 何金鑫¹ , 霍乃蕊¹ , 裴彩霞² , 古少鹏¹

1. 山西农业大学动物医学学院, 太谷 030801;
2. 山西农业大学动物科学学院, 太谷 030801

收稿日期: 2020-08-01

基金项目: 山西省重点研发计划项目(201803D221026-3)；山西省"1331工程"重点创新团队建设计划；山西省现代农业产业体系羊体系

作者简介: 韩郭皓(1997-), 男, 山西太原人, 硕士研究生, 从事兽医公共卫生与动物疾病防治研究。E-mail: hanguohao5@163.com.

通信作者: 古少鹏, 教授, 硕士生导师, E-mail: shpgu@163.com.

摘要: 本试验通过逐步递增饲粮精粗比诱导绵羊发生亚急性瘤胃酸中毒(SARA)，探究其对瘤胃异常代谢产物、瘤胃菌群、血气和血清生化指标的影响，为防治绵羊SARA提供理论依据。选取50只2月龄同期断奶健康的杜泊羊(♂)×湖羊(♀)F1代，随机分为2组。对照组(CK组，n=15)饲喂精粗比为50：50的基础饲粮；SARA诱导组通过饲粮精粗比递增诱导绵羊发生SARA，饲粮精粗比依次为50：50、70：30、80：20、90：10和100：0，各精粗比饲粮均饲喂7 d，瘤胃液pH在5.2~5.6持续3 h以上的绵羊构成SARA模型组。分别于各精粗比饲粮第7天晨饲后1、2、3、4、5、6和8 h采集瘤胃液，测定瘤胃异常代谢产物含量和瘤胃菌群结构。SARA诱导成功后，采集CK组和SARA模型组颈静脉血液，测定血气和血清生化指标。结果显示：1)在饲粮精粗比为100：0时，SARA诱导组中有27只绵羊瘤胃液pH在5.2~5.6持续3 h以上，部分羊腹泻，瘤胃液偏黄恶臭黏稠，符合SARA标准，构成SARA模型组。2)SARA模型组Chao1指数显著低于CK组(P＜0.05)，瘤胃菌群结构发生变化，乳酸产生菌相对丰度增加，瘤胃菌群碳水化合物代谢相关基因富集程度增加。3)SARA模型组瘤胃液和血液中乳酸、组胺和内毒素含量均显著高于CK组(P＜0.05)。4)SARA模型组血液二氧化碳总量、实际碳酸氢盐和全血碱剩余含量显著低于CK组(P＜0.05)。5)SARA模型组血清谷丙转氨酶活性显著低于CK组(P＜0.05)，血清生化指标处于正常范围。综上所述，绵羊SARA发生过程中，瘤胃菌群丰富度降低，菌群结构改变，乳酸产生菌相对丰度增加，血液和瘤胃液乳酸、内毒素和组胺含量升高，机体发生代谢性酸中毒。

关键词: 亚急性瘤胃酸中毒绵羊瘤胃菌群代谢性酸中毒炎症反应

Effects of Subacute Ruminal Acidosis on Rumen Abnormal Metabolites, Rumen Bacterial Flora and Blood Related Indexes of Sheep

HAN Guohao¹ , GAO Xiaosha¹ , DUAN Jinwei¹ , ZHANG Huiqin¹ , ZHENG Yan¹ , MA Linfeng¹ , HE Jinxing¹ , HUO Nairui¹ , PEI Caixia² , GU Shaopeng¹

1. College of Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;
2. College of Animal Science, Shanxi Agricultural University, Taigu 030801, China

Corresponding author: GU Shaopeng. E-mail: shpgu@163.com.

Abstract: In order to provide a theoretical basis for prevention and cure of sheep subacute ruminal acidosis (SARA), the effects of rumen abnormal metabolites, rumen bacterial flora, blood gas and serum biochemical indexes were explored after sheep SARA-induced by gradually increasing the concentrate to forage ratio. Fifty 2-month-old weaned Dorper sheep (♂)×Hu sheep (♀) F1 generations were randomly divided into two groups. The control group (CK group, n=15) was fed a basal diet with concentrate to forage ratio of 50:50, while the SARA induction groups (n=35) were fed diets with gradually increased the concentrate to forage ratios of 50:50, 70:30, 80:20, 90:10 and 100:0 for 7 d at each ratio. Sheep in the SARA induction groups whose rumen fluid pH was between 5.2 and 5.6 for more than 3 h constituted the SARA model group. The rumen fluid was collected at 1, 2, 3, 4, 5, 6 and 8 h after morning feeding on the 7th day of each ratio, and the rumen abnormal metabolites contents and bacterial flora structure were measured. Then, the jugular vein blood was collected from the CK group and the SARA model group, and the blood gas and serum biochemical indexes were measured. The results showed as follows: 1) when the concentrate to forage ratio was 100:0, rumen pH of 27 sheep in SARA induction group was 5.2 to 5.6 and lasted for more than 3 h, which were made into the SARA model group. Furthermore, some sheep had diarrhoea, and the rumen fluid was yellowish, smelly and sticky. 2) The Chao1 index of SARA model group was significantly lower than that of CK group (P < 0.05), the rumen bacterial flora structure changed, the relative abundance of lactic acid-producing bacteria increased, and the genes related to carbohydrate metabolism in the rumen bacterial flora increased. 3) The contents of lactic acid, histamine and endotoxin in the rumen fluid and blood of SARA model group were significantly higher than those of CK group (P < 0.05). 4) The contents of total amount of carbon dioxide, actual bicarbonate and total blood alkali in blood of SARA model group were significantly lower than those of CK group (P < 0.05). 5) The serum alanine aminotransferase activity of SARA model group was significantly lower than that of CK group (P < 0.05), and both serum biochemical indexes were in the normal range. In summary, the richness of rumen bacterial flora decreases, the bacterial flora structure changes and the lactic acid-producing bacteria relative abundance increases in SARA sheep, the contents of lactic acid, endotoxin and histamine in blood and rumen fluid increase, and metabolic acidosis occurs.

Key words: subacute ruminal acidosis sheep rumen bacterial flora metabolic acidosis inflammatory response

亚急性瘤胃酸中毒(subacute ruminal acidosis，SARA)是高产反刍动物群体中常见的营养代谢性疾病，绵羊育肥养殖过程中，为达最大生产性能常饲喂大量高精饲粮，高精饲粮发酵导致瘤胃内挥发性脂肪酸以及乳酸等有机酸积累，瘤胃pH长时间处于较低水平，绵羊发生SARA^[1]。SARA会导致瘤胃液pH下降、瘤胃微生物菌群紊乱、机体组织器官产生炎症、生产性能降低、动物淘汰或死亡等情况发生^[2-4]。SARA早期通常没有典型症状，诊断较为困难，瘤胃液pH 5.2~5.6持续3 h以上为SARA主要判定依据^[5]。SARA发生机制包括乳酸中毒学说以及内毒素组胺中毒学说^[6]。研究报道，奶牛SARA可引起瘤胃液乳酸、组胺和内毒素含量显著增加，但血液乳酸、组胺和内毒素含量变化不显著^[7-8]；而曾光^[9]通过梯度增加精粗比诱导山羊SARA，发现瘤胃液及血液乳酸、组胺和内毒素含量均显著升高。瘤胃内环境失衡通常会导致瘤胃菌群发生改变，Kim等^[10]饲喂高谷物饲粮诱导奶牛SARA，显示瘤胃菌群多样性指数下降，普雷沃氏菌属的相对丰度降低。目前，关于SARA的发生及其机制研究多集中于奶牛^[11-13]和山羊^[14-16]，模型多以短期高谷物饲粮诱导，诱导过程与疾病发生发展不符，SARA绵羊瘤胃异常代谢产物及菌群、血气和血清生化指标等如何变化鲜有报道，其发生机制尚不清楚。因此，本研究通过逐步提升饲粮精粗比建立绵羊SARA模型，分析SARA对绵羊瘤胃异常代谢产物、瘤胃菌群、血气和血清生化指标的影响，探究绵羊SARA状态下瘤胃内环境和机体状况，为防治绵羊SARA提供理论依据。

1 材料与方法 1.1 试验动物

选择50只2月龄同期断奶、平均体重(20.50±2.21) kg杜泊羊(♂)×湖羊(♀)F1代作为试验动物，公母各占1/2。

1.2 动物设计

试验前绵羊分为2组，分别为SARA诱导组(n=35)与对照组(CK组，n=15)。预试期30 d，饲喂精粗比为50 ∶ 50的基础饲粮。SARA诱导组通过饲粮精粗比递增诱导绵羊发生SARA，饲粮精粗比依次为50 ∶ 50、70 ∶ 30、80 ∶ 20、90 ∶ 10和100 ∶ 0，各精粗比饲粮均饲喂7 d，瘤胃液pH 5.2~5.6持续3 h以上的绵羊构成SARA模型组；CK组饲喂精粗比为50 ∶ 50的基础饲粮。每日06:00和17:00饲喂，每只羊每日约采食1.55 kg饲粮。饲粮参照《肉羊饲养标准》(NY/T 816—2004)配制，各精粗比饲粮组成及营养水平见表 1。

表 1 各精粗比饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of each concentrate to forage ratio diets (air-dry basis)

%
项目 Items	精粗比 Concentrate to forage ratio
项目 Items	50∶50	70∶30	80∶20	90∶10	100∶0
原料 Ingredients
花生秧 Peanut vine	50.00	30.00	20.00	10.00
玉米 Corn	30.00	50.00	60.00	70.00	80.00
麸皮 Wheat bran	5.50	5.50	5.50	5.50	5.50
豆粕 Soybean meal	7.50	7.50	7.50	7.50	7.50
豆饼 Soybean cake	4.00	4.00	4.00	4.00	4.00
预混料 Premix¹⁾	2.50	2.50	2.50	2.50	2.50
碳酸氢钠 NaHCO₃	0.50	0.50	0.50	0.50	0.50
合计 Total	100.00	100.00	100.00	100.00	100.00
营养水平 Nutrient levels²⁾
代谢能 ME/(MJ/kg)	10.10	10.91	11.29	11.81	12.15
干物质 DM	86.04	85.65	85.48	85.22	85.06
粗蛋白质 CP	13.41	12.67	12.32	11.95	11.56
非纤维性碳水化合物 NFC	40.35	46.25	49.86	54.22	59.18
中性洗涤纤维 NDF	39.63	25.32	20.85	16.13	13.32
酸性洗涤纤维 ADF	20.78	14.43	10.93	8.03	5.78
钙 Ca	0.56	0.52	0.49	0.47	0.45
磷 P	0.23	0.24	0.26	0.25	0.26
钙磷比 Ca/P	2.43	2.17	1.88	1.88	1.65
1)每千克预混料含有One kilogram of premix contained the following：VA 200 000 IU，VD₃ 50 000 IU，VE 750 mg，Fe 1.5 g，Cu 250 mg，Zn 1.48 g，Mn 990 mg，I 15 mg，Se 15 mg，Co 10 mg。 2)代谢能为计算值，其他均为实测值。ME was a calculated value, while the others were measured values.

表 1 各精粗比饲粮组成及营养水平(风干基础) Table 1 Composition and nutrient levels of each concentrate to forage ratio diets (air-dry basis)

1.3 临床观察与样品采集测定 1.3.1 临床观察

每日观察绵羊精神和粪便状态，采集瘤胃液时观察其颜色、气味和黏稠度，并记录。

1.3.2 样品采集

于各饲喂期第7天晨饲后1、2、3、4、5、6和8 h使用瘤胃液口腔采样器(A1164K，武汉科立博牧业科技有限公司)抽取瘤胃液，每只50 mL，4层纱布过滤保存。模型构建成功后，于晨饲前用BD Preset^TM采血器和含促凝剂的采血管采集颈静脉血液。

1.3.3 样品测定

瘤胃液采集后，立即用便携pH计(AS-PH8，武汉中测宏图测量仪器有限公司)测定。随机选取3只羊同一天的瘤胃液样品混匀后用于菌群测定，5个混匀瘤胃液样本用于瘤胃异常代谢产物测定，5个血液样本用于血气和血清生化指标测定。瘤胃液菌群16S rRNA V3~V4区测序由天津诺禾致源公司完成。乳酸、组胺和内毒素含量分别使用南京建成生物工程研究所、江苏酶免实业有限公司和厦门鲎试剂生物科技股份有限公司生产的试剂盒测定。血液pH、二氧化碳分压(pCO₂)、氧分压(pO₂)、二氧化碳总量(tCO₂)以及乳酸、实际碳酸氢盐(HCO₃^-)、阴离子间隙(AG)和全血碱剩余(BE-B)含量使用全自动血气分析仪(GEM Premier 4000，美国实验室仪器公司)检测。血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)活性及尿素(UN)、总胆红素(TBIL)、直接胆红素(DBIL)、间接胆红素(IBIL)、总蛋白(TP)、白蛋白(ALB)、球蛋白(GLB)和葡萄糖(GLU)含量使用全自动生化分析仪(TBA-120 FR，日本)检测。

1.4 数据分析

测序数据使用Qiime 1.9.1软件进行处理分析。有效测序数据以97%的一致性聚类成为操作分类单元(operational taxonomic units，OTUs)，使用SILVA 132数据库进行物种注释。Alpha分析使用wilcox秩和检验进行统计分析。数据使用Excel 2019整理，统计分析应用SPSS 26.0软件中独立样本t检验及单因素方差分析，多重比较采用Duncan氏法。数据以平均值±标准差(mean±SD)表示。

2 结果 2.1 SARA绵羊模型建立

由表 2可见，SARA诱导组瘤胃液pH随饲粮精粗比提高而降低，精粗比为100 ∶ 0时共有27只绵羊瘤胃液pH在5.2~5.6持续3 h以上。临床观察，CK组绵羊未见明显异常，瘤胃液较稀薄，气味微臭，色泽偏绿(图 1-A)，粪便为较硬颗粒状(图 1-C)；SARA诱导组在精粗比为100 ∶ 0时部分绵羊瘤胃液较浓稠，气味恶臭，色泽偏黄(图 1-B)，轻度腹泻，粪便稀软不成形(图 1-D)，精神状态未见明显异常。综上判定，SARA诱导组的27只绵羊在精粗比为100 ∶ 0时发生SARA，构成SARA模型组^[5]。

表 2 SARA对绵羊瘤胃液pH的影响 Table 2 Effects of SARA on rumen fluid pH of sheep

项目 Items	精粗比 Concentrate to forage ratio
	SARA诱导组 SARA induction group					对照组 CK group
	50∶50	70∶30	80∶20	90∶10	100∶0	50∶50
瘤胃液 pH Rumen fluid pH
平均值 Average value	6.62±0.19^a	6.40±0.13^a	6.10±0.22^b	5.70±0.23^c	5.43±0.19^d	6.45±0.14^a
最大值 Maximum value	6.72±0.07^a	6.52±0.05^c	6.34±0.05^d	5.84±0.06^e	5.68±0.05^f	6.64±0.06^b
最小值 Minimum value	6.46±0.06^a	6.26±0.04^b	5.83±0.04^c	5.42±0.10^d	5.24±0.03^e	6.27±0.05^b
持续时间 Duration time/(h/d)
pH < 5.6	0	0	0	1.82±0.67^b	5.33±1.11^a	0
pH < 5.2	0	0	0	0	0	0
同行数据肩标不同小写字母表示差异显著(P＜0.05)，相同或无字母表示差异不显著(P>0.05)。下表同。 In the same row, values with different small letter superscripts mean significant difference (P＜0.05), while with the same or no letter superscripts mean no significant difference (P>0.05). The same as below.

表 2 SARA对绵羊瘤胃液pH的影响 Table 2 Effects of SARA on rumen fluid pH of sheep

A：健康绵羊瘤胃液rumen fluid of healthy sheep；B：SARA绵羊瘤胃液rumen fluid of SARA sheep；C：健康绵羊粪便feces of healthy sheep；D：SARA绵羊粪便feces of SARA sheep。图 1 SARA对绵羊瘤胃液及粪便的影响 Fig. 1 Effects of SARA on rumen fluid and feces of sheep

2.2 SARA对绵羊瘤胃液乳酸、组胺和内毒素含量的影响

由表 3可见，瘤胃液乳酸、组胺和内毒素含量随饲粮精粗比提高而增加，SARA模型组瘤胃液乳酸、组胺和内毒素含量均显著高于CK组(P＜0.05)。

表 3 SARA对绵羊瘤胃液乳酸、组胺和内毒素含量的影响 Table 3 Effects of SARA on contents of lactic acid, histamine and endotoxin in rumen fluid of sheep

2.3 SARA对绵羊瘤胃菌群的影响 2.3.1 Alpha多样性分析

由表 4可见，随饲粮精粗比提高，瘤胃菌群丰富度指数(Chao1指数)和多样性指数(Shannon指数)下降。SARA模型组Chao1指数显著低于CK组(P＜0.05)。

表 4 SARA对绵羊瘤胃菌群Alpha多样性的影响 Table 4 Effects of SARA on Alpha diversity of rumen bacterial flora of sheep

2.3.2 Beta多样性分析

非度量多维标度(non-metric multidimensional scaling，NMDS)分析基于Weighted Unifrac距离，SARA模型组位于第1及第4象限，与CK组距离较远(图 2-A)；主坐标分析(principal coordinates analysis，PCoA)基于Bray Curtis距离，获得主坐标1(PC1)的贡献率为17.14%，SARA模型组位于第4象限，样本较为集中，与CK组距离较远(图 2-B)。

A：非度量多维标度分析NMDS analysis；B：主坐标分析PCoA。图 2 SARA对绵羊瘤胃菌群Beta多样性的影响 Fig. 2 Effects of SARA on Beta diversity of rumen bacterial flora of sheep

2.3.3 门水平与属水平下瘤胃菌群结构分析

门水平下，SARA诱导组在精粗比为90 ∶ 10时最优菌门为厚壁菌门(Firmicutes)，其余各组最优菌门为拟杆菌门(Bacteroidetes)；与CK组相比，SARA模型组厚壁菌门、放线菌门(Actinobacteria)和变形菌门(Proteobacteria)相对丰度增加，拟杆菌门相对丰度降低(图 3-A)。属水平下，SARA诱导组在精粗比为90 ∶ 10时最优菌属为Pseudoscardovia，其余各组最优菌属为普雷沃氏菌科未确定菌属(unidentified Prevotellaceae)；与CK组相比，SARA模型组普雷沃氏菌科未确定菌属、罗氏菌属(Roseburia)和Pseudoscardovia相对丰度增加(图 3-B)。SARA模型组在属水平下，双歧杆菌属(Bifidobacterium)和Pseudoscardovia为具有生物学及统计学意义的差异物种(图 3-C)。

A：门水平菌群结构堆积柱状图stacking histogram of bacterial community structure at phylum level；B：属水平菌群结构堆积柱状图stacking histogram of bacterial community structure at genus level；C：线性判别分析分枝进化图LEfSe analysis branching evolution diagram。图 3 SARA对绵羊瘤胃菌群结构的影响 Fig. 3 Effects of SARA on rumen bacterial flora structure of sheep

2.3.4 Tax4Fun功能预测

由图 4可见，SARA模型组瘤胃菌群代谢相关二级功能层基因富集存在差异，与CK组相比，主要体现在碳水化合物代谢相关基因的相对丰度升高，辅助因子和维生素的代谢、脂质代谢、其他次生代谢产物的生物合成和其他氨基酸的代谢相关基因的相对丰度降低。

图 4 代谢相关功能基因预测热图 Fig. 4 Heatmap of metabolism-related functional gene prediction

2.4 SARA对绵羊血气分析的影响

由表 5可见，血气分析结果显示，SARA模型组血液乳酸含量显著高于CK组(P＜0.05)，SARA模型组血液二氧化碳总量、实际碳酸氢盐及全血碱剩余含量均显著低于CK组(P＜0.05)。

表 5 SARA对绵羊血气分析的影响 Table 5 Effects of SARA on blood gas analysis of sheep

2.5 SARA对绵羊血清生化指标的影响

由表 6可见，SARA模型组血清组胺和内毒素含量显著高于CK组(P＜0.05)，血清谷丙转氨酶活性显著低于CK组(P＜0.05)，其余血清生化指标与CK组差异不显著(P>0.05)。

表 6 SARA对绵羊血清生化指标的影响 Table 6 Effects of SARA on serum biochemical indexes of sheep

3 讨论

瘤胃pH受饲粮影响，一般情况下，反刍动物瘤胃pH维持在6.5~7.5^[17]。瘤胃菌群变化与饲粮组成、饲养环境和管理模式等相关^[18-19]。动物长期采食高精料饲粮，瘤胃异常发酵，导致瘤胃pH下降，瘤胃菌群改变^[20]。Plaizier等^[21]诱导奶牛SARA，发现瘤胃微生物区系丰富度和多样性降低。众多研究发现，反刍动物瘤胃最优菌门为拟杆菌门，最优菌属为普雷沃氏菌属^[22-27]。李小玉等^[27]研究急性瘤胃酸中毒山羊，发现瘤胃厚壁菌门相对丰度增加，拟杆菌门相对丰度降低。研究表明，SARA奶牛普雷沃氏菌属相对丰度提升^[28]，瘤胃壁菌群碳水化合物代谢相关基因高表达^[29]。本研究与上述结果相似，瘤胃内酸性环境导致瘤胃菌群发生变化，大量瘤胃细菌因不适酸性环境而死亡，进而菌群丰富度和多样性降低，造成瘤胃健康破坏和生产效能降低^[30]。SARA模型组瘤胃厚壁菌门相对丰度增加，主要为降解纤维类物质和碳水化合物的菌种，进而引起菌群碳水化合物相关代谢基因富集，有助于反刍动物瘤胃代谢^[24]。绵羊发生SARA后，瘤胃内毒素含量显著升高，因拟杆菌门内革兰氏阴性细菌大量死亡，拟杆菌门相对丰度降低，细菌裂解释放内毒素^[25]。罗氏菌属相对丰度提升，瘤胃液乳酸含量显著增加，表明SARA会增加瘤胃乳酸产生菌，导致瘤胃乳酸积累，pH下降。

SARA模型组血气分析结果与胡红莲^[14]和苑学^[31]研究结果一致，血液pH、二氧化碳分压、实际碳酸氢盐和全血碱剩余含量降低，氧分压含量升高。SARA绵羊血液乳酸含量显著升高，表明瘤胃液乳酸吸收超过肝脏处理能力，导致血液pH下降，血液过多的氢离子(H⁺)由血液实际碳酸氢盐缓冲，进而降低机体碱储，发生代谢性酸中毒。SARA模型组瘤胃内毒素积累，促进炎症介质组胺的产生，二者同时进入血液循环，血液与瘤胃液内毒素和组胺含量显著升高，可能会引发一系列炎症反应^[32]。血清生化指标可以反映机体营养代谢状况以及肝肾功能状况。孙燕勇等^[33]研究发现，SARA山羊有一定程度的肝功能损伤，本研究未发现肝脏损害，各项血清生化指标均处于正常范围，可能与SARA持续时间有关。

4 结论

本研究通过逐步递增饲粮精粗比成功构建绵羊SARA模型。绵羊发生SARA时，瘤胃内环境改变，瘤胃菌群丰富度降低，厚壁菌门、放线菌门、变形菌门、普雷沃氏菌科未确定菌属、罗氏菌属和Pseudoscardovia相对丰度增加，拟杆菌门相对丰度下降，血液和瘤胃液乳酸、内毒素和组胺含量升高，机体碱储降低，发生代谢性酸中毒。

致谢:

感谢山西保森畜牧有限公司提供的试验场地。

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