【作者】 熊江林；

【导师】 刘建新；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2014， 博士

【摘要】 针对当前奶牛养殖过程中奶牛采食黄曲霉毒素B1(Aflatoxin B1.AFB1)污染饲料带来的牛奶和奶制品黄曲霉毒素M1(Aflatoxin Ml, AFM1)的污染问题,本文研究了日粮中AFB1到牛奶的转化率、牛奶中AFM1的清除规律,以及AFBl对奶牛生产性能和健康的影响。同时,评价了吸附剂Solis Mos (SM)的添加效果。具体研究内容与获得的结果如下。1、长三角地区奶牛场日粮中AFB1和原料奶AFM1的季节性变化(试验一)本试验调研了长三角地区18家奶牛场全混合日粮(Total Mixed Ration, TMR)中AFB1和原料奶AFM1在4个季节的发生率和污染程度。每个季节收集15份TMR样本,4个季节共采集60份TMR样本；而每个季节从奶缸中采集18份混合牛奶样,4个季节共采集72份原料奶样。TMR中AFB1测定应用HPLC-FLD方法(检出限为0.3μg/kg),牛奶AFM1测定应用LC-MS/MS方法(检出限为0.01μg/L)。应用SAS9.2(SAS Institute Inc., Cary, NC)单因素方差分析程序检验4个季节的TMR AFB1和牛奶AFM1的浓度差异。AFB1在28份TMR样本(46.7%)中被检出,AFB1浓度范围0.332-4.040ug/kg。冬季TMR中AFB1浓度显著高于春季和秋季(P<0.05),但与夏季没有显著差别。总共72份奶样中有43份牛奶样检测出AFM1(59.7%),其浓度范围为0.01-0.42μg/L。冬季原料奶中AFM1浓度(0.123μg/L)显著高于其他季节,春季(0.029μg/L)、夏季(0.032μg/L)与秋季(0.032μg/L)牛奶样AFM1浓度差异不显著(P>0.05)。本试验结果显示,TMR中AFB1以及牛奶中AFM1的污染呈现季节性差异,冬季和夏季饲料易致AFB1污染,而冬季牛奶污染AFM1风险最高,因此对牧场饲料和牛奶的AFs管理中需要考虑季节性因素。2.日粮AFB1向牛奶的转化以及日粮中添加SM的效果(试验二)本研究的目的是研究日粮AFB1到乳AFM1的转化率和乳中AFM1的清除规律,并评价SM对摄入不同剂量AFB1的奶牛乳中AFM1、奶牛血液生化指标和瘤胃发酵参数的影响。选择24头泌乳后期经产荷斯坦奶牛(泌乳天数271±29d；奶产量21.6±3.1kg/d),随机分为3组,每组8头。3组奶牛被安排进行3个小试,每个小试含2个处理,每个处理4头牛,采用交叉试验设计。小试1、2、3中奶牛分别采食含AFB1含量为0、20、40μg/kg.DM的日粮,每个小试中,奶牛接受对照组或添加0.25%的SM处理组。每个小试分为两个连续的时期,每期含有4d预试期(d1-4)、7d AFB1饲喂期(d5-11)和5d清除期(d12-16)。完成第一期试验后,将对照组和处理组对换,进入第二期试验。在每期试验第1、2和10-14d,采集TMR和牛奶样本；d1、11和14的晨饲前,通过尾静脉采集10mL血液至肝素管；d1和11晨饲后2h,通过口腔瘤胃导管采集50mL瘤胃液。结果发现,采食基础日粮奶牛乳中AFM1的本底水平是0.003gg/L；采食含20μg/kg AFB1日粮的奶牛乳中AFM1均值为0.105μg/L,日粮AFB1到牛奶的转化率为0.56%；采食含40μg/kg AFB1日粮的奶牛乳中AFM1均值为0.209μg/L,日粮AFB1到牛奶的转化率为0.59%。当奶牛停止采食AFB1污染日粮3d后,乳中AFM1即可清除。添加SM到含20μg/kg AFB1的日粮中能够显著减少奶中AFM1浓度(0.105vs.0.088μg/L, P<0.05),降低AFB1向牛奶中的转化率(0.56vs.0.46%,P<0.05)；但在含40μg/kg AFB1日粮中添加SM并不能降低牛奶中AFM1浓度。在基础日粮中添加SM能够增加总挥发性脂肪酸(VFA)浓度(99.6vs.94.2mM, P<0.05)、微生物蛋白(MCP)含量(3.3vs.2.9mg/mL, P<0.05)以及羧甲基纤维素酶(CMCase)活性(0.34vs.0.26U, P<0.05);在含20μg/kgAFB1日粮中添加SM能增加总VFA浓度(99.8vs.93.4mM, P<0.05), MCP含量(3.2vs.2.7mg/mL, P<0.05)以及CMCase活性(0.28vs.0.22U, P=0.07);但是,在含40μg/kg AFB1日粮中添加SM并未改变瘤胃发酵参数。无论是在基础日粮,还是在AFB1污染日粮中添加0.25%的SM,均不影响奶牛泌乳性能、肝功能和体液免疫,但可降低机体氧化应激。本试验结果表明,日粮中AFB1转化为乳AFM1的比率为0.46%-0.59%,乳中AFM1彻底清除需要3d；添加SM不影响奶牛的产奶量和乳成分；在20μg/kgAFB1日粮中添加SM可降低奶牛AFM1浓度,改善奶牛健康状况。3.长期摄入AFB1对奶牛生产性能和健康的影响以及SM的添加效果(试验三)本研究的目的是评估长期摄入AFB1的奶牛泌乳性能和健康状况,以及添加霉菌毒素吸附剂SM的效果。40头泌乳初期荷斯坦奶牛根据泌乳天数(33±7d)和奶产量(33.9±3.1kg)随机分为4个处理组。采用2因子2水平交叉试验设计,即AFB1(0或20μg/kg DM)和添加SM(0或0.25%)。奶产量和乳成分每周测定,血清生化指标和瘤胃发酵参数在正试期前1天和试验第8周进行测定。乳中AFM1采用HPLC-MS/MS进行分析。应用SAS程序的MIXED模型对试验数据进行统计分析。添加AFB1或SM不影响干物质采食量(23.9±0.34kg/d)、产奶量(35.5±0.76kg/d)、乳蛋白(2.92±0.056%)、乳脂肪(3.64±0.108%)和线性体细胞(5.1±0.39)等泌乳性能。在含AFB1的日粮中添加SM能显著降低乳中AFM1浓度(0.191vs.0.134μg/L, P<0.01)和AFB1转化为乳AFMl的比率(1.38vs.0.89%,)。给奶牛饲喂AFB1污染饲料会降低过氧化物岐化酶、总抗氧化能力、谷胱甘肽过氧化酶活性、IgG和IgA含量(P<0.05),而增加丙二醛浓度(P<0.05)。日粮中添加SM能够增加血浆过氧化物岐化酶活性、总抗氧化能力和IgG的含量,而减少丙二醛含量(P<0.05)。 AFB1和SM均不影响血浆谷丙转氨酶、谷草转氨酶、碱性磷酸酶活力和IgM浓度(P>0.05)。AFB1仅降低CMCase活性而不影响其它发酵参数。然而,添加SM可以增加NH3-N、 MCP和VFA含量和CMCase活性、降低pH值,但并不影响VFA组成以及木糖酶和淀粉活性。本试验结果表明,日粮中AFB1分泌到泌乳早、中期奶牛乳中的比率高于后期；AFB1或SM均不影响奶产量和乳成分,但添加SM可降低乳中AFM1浓度,减缓长期摄入AFB1给奶牛健康带来的不利影响。综上所述,牧场乳品安全需要考虑牛奶和饲料AFs污染的季节性差异；泌乳早中期和后期奶牛将日粮AFB1转化乳AFM1的比率分别为0.89%-1.38%和0.46%-0.59%；本试验条件下,SM或AFB1均不影响泌乳奶牛的产奶量和乳成分；添加SM可降低AFB1的转化率,改善奶牛的健康状况。更多还原

【Abstract】 In the current dairy farming, dairy cows ingest aflatoxin B1(AFB1)-contaminated feed, which often causes aflatoxin M1(AFMl) contamination in milk and milk products. This study was conducted to investigate:(1) transfer rate of dietary AFB1into milk;(2) Clearance pattern in milk AFM1after removal of AFB1from diet;(3) the effect of AFB1fed on productive performance and healthy condition in dairy cows; and (4) the effect of dietary inclusion of SM on the above-mentioned variables. The experiments and main results obtained are summarized as below.1. Seasonal variation of aflatoxin Bl in feed and aflatoxin Ml in raw milk in Yangtze River Delta region of China (Exp.1)The objective of the study was to evaluate the occurrence of AFB1in total mixed ration (TMR) and AFM1in raw milk from18dairy farms in Yangtze River Delta region in four seasons. A total of60TMR samples were collected with15samples each season, and72milk tank samples were collected with18samples each season. Feed AFB1was tested using HPLC-FLD and milk AFM1was detected using LC-MS/MS method. The differences in the concentration of feed AFB1and milk AFMl among four seasons were analyzed with a one-way ANOVA procedure. With detection limit of0.300μg/kg, AFB1was found to be positive in28TMR samples (46.7%) ranging from0.332to4.040μg/kg. A higher AFB1concentration in TMR was observed in winter than that in spring and autumn (P<0.05), with no difference with summer. Milk AFM1was found to be positive in43milk samples (59.7%) ranging from0.01to0.42μg/L with detection limit of0.01μg/L. The concentration of AFM1in raw milk was significantly higher during the winter (0.123μg/L) than that in other seasons (P<0.05). There was no significant difference among the spring (0.029μg/L), summer (0.032μg/L), and autumn (0.032μg/L)(P>0.05) seasons. These result indicated that there is large variation in the occurrence of feed AFB1and milk AFMl during different seasons. The feed in winter and summer is in high risk for AFB1, while the milk in winter is in high risk for AFM1, suggesting that seasonal factors should be considered for control of aflatoxin in both the feed and milk. 2. Transfer of dietary AFB1to milk AFM1and effect of dietary inclusion of SM in dairy cows (Exp.2)The objectives of this study was to investigate the transfer rate of AFB1from feed to milk and clearance pattern of milk AFM1after termination of dietary AFB1administration, and to evaluate the effects of SM on milk AFM1, plasma biochemical variables and ruminal fermentation of dairy cows fed varying doses of AFB1. Three equal groups of8multiparous Holstein cows in late lactation (DIM=271±29d; Milk yield=21.6±3.1kg/d) were assigned to one of three trials in a crossover design. Cows in Trial1received no aflatoxin, cows in Trial2received20μg of AFB1/kg of diet DM, and cows in Trial3received40μg of AFB1/kg of diet DM. Cows in each trial were assigned to1of2treatments:control or0.25%SM. Each trial consisted of2consecutive periods with pre-trial for4d (d1to4), AFB1challenging for7d (d5toll) and clearing for5d (d12to16). In each period, samples of TMR and milk were collected on d1,2and10to14of each period. Blood samples (10mL) were collected from the coccygeal vein into heparinized vacuum tubes before the morning feeding on d1,11and14. Rumen fluid (50mL) was collected by oral stomach tube2h after the morning feeding on d1and11. Background level of milk AFM1was0.003μg/L. When cows were fed with20or40μg/kg AFB1-contaminated diets, average contents of milk AFM1were0.105and0.209μg/L, respectively, and transfer rates of dietary AFB1into milk were0.56and0.59%, respectively. Milk AFM1could be thoroughly cleared3days after dairy cows were fed AFB1-free diet. Adding SM in the20μg/kg of AFB1-contaminated diet decreased the milk AFM1concentration (0.105vs.0.088μg/L) and the transfer of aflatoxin to milk (0.56vs.0.46%), but they were not affected by addition of SM in the40μg/kg of AFB1-contaminated diet. Addition of SM to the basal diet increased overall ruminal concentrations of volatile fatty acid (VFA,99.6vs.94.2mM, P<0.05), microbial cell protein (MCP,3.3vs.2.9mg/mL, P<0.05) and carboxymethy cellulase (CMCase) activity (0.34vs.0.26U, P<0.05). Adding SM in the20μg/kg of AFB1-contaminated diet increased the total VFA concentration (99.8vs.93.4mM, P<0.05), MCP level (3.2vs.2.7mg/mL, P<0.05) and CMCase activity (0.28vs.0.22U, P=0.07), but they were not affected by addition of SM in the40μg/kg of AFB1-contaminated diet. Adding SM to basal or AFB1-contaminated diets at0.25%(DM basis) had no effect on lactation performance, liver function and humoral immunity, but significantly decreased indicators of body oxidative stress. From the results obtained in this experiment, it is concluded that transfer rate of dietary AFB1 into milk AFM1ranged from0.46to0.59%and milk AFM1can be cleaned within3d; adding SM did not affect milk yield and compositon of dairy cows in late lactation, but decreased milk AFM1concentration and improved healthy condition in dairy cows fed the diet contaminated with20μg/kg AFB1.3. Effects of long-term challenge of AFB1on productive performance and healthy condition in dairy cows (Exp.3)The objective of the study was to evaluate produtive performance and healthy condition in dairy cows exposed to long-term challenge of AFB1, and to study the effect of SM inclusion. Forty dairy cows were blocked into4groups based on days in milk (33±7; mean±SD) and milk production (33.9±3.1kg; mean±SD), and were randomLy assigned to1of4treatments in a2×2factorial arrangements with AFB1(0or20μg/kgDM) and SM (0or0.25%of DM) as main factors. The experiment lasted for9weeks, with the first week for adaptation. Milk yield and milk composition were recorded weekly, and plasma biochemical variables and ruminal fermentation were analyzed in the first and the last week of the experiment. Milk AFM1was analyzed by HPLC-MS/MS. Variables of data were analyzed using the mixed procedure of SAS. Dry matter intake, milk yield, contents of milk protein and milk fat, and linear somatic cell count averaged23.9kg/d,35.5kg/d,2.9%,3.6%, and5.1, respectively, and were not affected (P>0.05) by either AFB1or SM addtion. Addition of SM in AFB1-contaminated diet significantly reduced (P<0.01) milk AFM1concentration (0.191vs.0.134μg/L) and transfer rates of dietary AFB1into milk AFM1(1.38vs.0.89%). Compared to the cows fed non-AFB1diet, Dairy cows fed AFB1-contaminated diet had lower level of superoxide dismutase activity, total antioxidative capacity, glutathione peroxidase, IgG and IgA (P<0.05), and higher level of malondialdehyde in plasma (P<0.05). Inclusion of SM into diets increased the plasma superoxide dismutase activity, total antioxidant capacity, and IgG, but decreased malondialdehyde (P<0.05). Neither AFB1nor SM affected the plasma levels of alanine transaminase, aspartate aminotransferase, total bilirubin, and alkaline phosphatase and IgM (P>0.05). The AFB1did not affect ruminal fermentation parameters, except for decreasing CMCase activity. However, addition of SM increased the level of ammonia-N, MCP, VFA and CMCase and decreased the pH value, but the individual VFA proportion, and other ruminal enzyme activity were not influenced by SM. It is indicated from this experiment that transfer rate of dietary AFB1into milk was higher than that for late-lactation cows; adding SM did not affect milk yield and compositon of dairy cows in early to mid lactation, but decreased milk AFM1level and improved adverse healty condition in dary cows exposed to long-term challenge of AFB1.In Summary, seasonal difference in TMR AFB1and milk AFM1should be considered for the safe milk production in the dairy farms of Yangtze River Delta region. The transfer rate of dietary AFB1into milk AFM1ranged from0.89to1.38%or0.46to0.59%in early-to-mid-or late lactating dairy cows, respectively. The AFM1can be cleaned within3d from the milk of dairy cow receiving an AFB1-contaminated diet. Adding SM or AFB1did not affect milk yield and compostion, while SM addtion decreased the transfer rate and improved healty condition in dairy cows.更多还原