饲料中洛克沙胂的固相萃取--高效液相色谱法检测技术研究

建立了固相萃取(SPE)——高效液相色谱(HPLC)法测定饲料中洛克沙胂的分析方法,优化了提取方法、净化参数、萃取容量、流速和洗脱溶剂等条件。饲料采用20g/l的磷酸氢二钾溶液作提取液,用阴离子交换柱萃取净化的方法;色谱条件为:在266nm波长下检测,色谱柱为SymmetryShieldTMRP18柱(150mm×4.6mm),流动相为含0.1%甲酸的0.05mol/l磷酸二氢钾溶液-甲醇(体积比95:5),流速为1ml/min,洛克沙胂的浓度在0.2～10.0mg/l范围内含量与峰面积呈良好的线性关系,相对标准偏差为0.5%~1.07%,回收率为87.5%~96.7%。饲料中洛克沙胂的定量检出限为5mg/kg。     

洛克沙胂对猪生产性能和血液指标的影响

本研究通过断奶仔猪、生长猪和肥育猪3个试验研究了日粮添加洛克沙胂对猪生产性能及血液指标的影响。试验分别选择96头杜×长×大三元杂交断奶仔猪(8.29±0.99)kg、生长猪(32.68±3.43)kg和肥育猪(62.74±8.89)kg,随机分为4个处理,每个处理6个重复,每个重复4头猪,公母各半。处理1为对照组,日粮中不添加洛克沙胂,处理2、3和4分别在日粮中添加50、100 mg/kg和150 mg/kg洛克沙胂。基础日粮为玉米-豆粕型,各处理日粮营养水平均为等能量、等蛋白。结果表明:日粮中添加洛克沙胂能显著提高断奶仔猪与生长猪日增重和饲料转化效率(P<0.05),减少仔猪腹泻率(P<0.01)。但对肥育猪增重效果不明显(P>0.05);日粮中添加洛克沙胂使动物血红蛋白、肌酐和谷草转氨酶含量升高,白蛋白含量降低(P<0.05)。表明洛克沙胂对猪具有一定的促生长效果,可增加动物血液运氧能力,但同时有可能对动物肝脏、肾脏造成一定损伤。     

饲料中阿散酸和洛克沙胂测定方法的研究

通过检测条件:提取液、净化、流动相组成、PH值优化,对饲料中阿散酸、洛克沙胂进行高效液相色谱测定,其检测限:阿散酸为2.0mg/kg,洛克沙胂为8.0mg/kg;检测回收率达81.9-99.2%,组内、组间变异系数为2.1-8.5%,符合分析要求。     

猪排泄物中恩诺沙星和环丙沙星含量的HPLC检测方法

为了对猪排泄物中恩诺沙星(enrofloxacin,ENR)和环丙沙星(ciprofloxacin,CIP)进行定量检测,试验建立了测定猪粪尿中ENR和CIP含量的高效液相－荧光检测方法。将猪粪经乙腈－氨水超声提取后,加入三氯乙酸酸化,然后分别将经磷酸酸化后的猪尿和提取后的猪粪溶液经固相萃取小柱富集净化,取净化液进行HPLC分析。HPLC流动相为乙腈(A):柠檬酸/乙酸铵缓冲液(B),梯度洗脱:0~25 min,A 10%~40%;25~30 min,A 40%至10%,荧光检测器的激发波长278 nm,发射波长465 nm。结果表明,ENR和CIP 在尿中的最低检测限(LOD)<0.01 mg/L,在粪中的LOD<0.021 mg/kg,在尿中的最低检测限(LOQ)<0.03 mg/L,在粪中LOQ<0.056 mg/kg,猪尿中的ENR和CIP在0.01~1.0 mg/mL范围内线性关系良好,R²分别为0.9994和0.9992;猪粪中的ENR和CIP在0.02~2.0 mg/mL范围内线性关系良好,R²分别为0.9986和0.9981。ENR在猪粪和猪尿中的回收率分别为79.4%和88.5%,CIP在猪粪和猪尿中的回收率分别为75.8%和89.9%。该方法样品处理简单,检测结果准确可靠,且灵敏度较高,是值得推广的检测方法。     

猪体恩诺沙星内服和肌注途径给药排泄规律的比较

给10头猪分别按体重5mg／kg单剂量内服和肌注恩诺沙星，采集自然排泄的猪粪、尿，用高效液相一荧光法测定恩诺沙星及其代谢产物环丙沙星浓度，计算采样间隔内单位时间药物排泄量占给药量的比例，比较2种不同给药途径原形药物和环丙沙星的排泄规律。结果显示，2种给药途径对恩诺沙星和环丙沙星总排泄量的影响不显著，仅对药物排泄量与时间之间的关系产生影响；2种给药途径的试验猪粪样中恩诺沙星的排泄量均高于尿样；内服与肌注给药后96h内，粪和尿中检出的环丙沙星分别占给药量的3．93％和4．02％，累积排泄的恩诺沙星和环丙沙星之和分别占给药量的9．89％（内服）和9．57％（肌注）。     

不同抗菌素对断奶仔猪生长性能的影响

本试验探讨断奶仔猪日粮中添加不同抗菌素(100mg/ kg磺胺二甲嘧啶 50mg/kg口服氨苄青霉素 100mg/ kg土霉素、100mg/ kg杆菌肽锌 20mg/kg硫酸抗敌素、100mg/kg硫酸新霉素、50mg/kg洛克沙胂 50mg/ kg喹乙醇、300mg/kg痢特灵)对断奶仔猪生长性能和腹泻的影响。试验结果表明,断奶仔猪日粮中添加抗菌素的日增重和日耗料与对照组相比无显著差异(P> 0.05);但添加 50mg/ kg洛克沙胂 50mg/ kg 喹乙醇组的饲料增重比明显优于对照组(P<0.05);添加抗菌素各组的腹泻情况均比对照组有所改善。从生长性能及腹泻情况综合考虑,断奶仔猪日粮中添加50mg/kg洛克沙胂 50mg/kg喹乙醇稍好于添加其它抗菌素。     

高效液相色谱法检测鸡粪便中洛克沙胂

建立了用固相萃取-高效液相色谱法(HPLC)测定鸡粪便中洛克沙胂(ROX)的方法。鸡粪便采用0.05 mol/L磷酸氢二钾∶甲醇=90∶10(V/V)溶液作提取液,用Waters Oasis(MAX固相萃取小柱净化;色谱条件为:Agilent C18色谱柱(4.6×250 mm,5μm),流动相为0.05 mol/L磷酸二氢钾∶100 mL/L乙酸∶甲醇=90∶7∶3(V/V/V),流速1.0 mL/min,柱温30℃,进样量20μL,紫外检测波长266 nm。ROX的浓度在10.0μg/mL范围内,线性相关系数为0.999 9,方法的回收率为80.09%～81.5%,变异系数为2.09%～5.87%,检出限为1.0 mg/kg。该方法精确度和灵敏度很高,适用于鸡粪便中ROX含量的测定。从某鸡场随机采集8个粪样,调查鸡粪中的ROX含量,其ROX的平均浓度为21.49 mg/kg。     

蛋氨酸铜对猪血清铜锌含量及粪铜锌排泄量的影响

为了研究饲粮中添加蛋氨酸铜与硫酸铜对生长猪生长性能、养分消化率、血清铜锌含量及粪铜辞排泄量的影响.选择体重为17.17±2.34kg(P＞0.05)的二元杂交(长×大)生长阉公猪30头进行试验,试猪按完全随机区组设计,依体重和窝别随机分为5个处理,每个处理3个重复,每个重复2头猪,分别饲喂10、150、250mg/kg蛋氨酸铜和150、250mg/kg硫酸铜,试验期30 d.结果表明:饲喂蛋氨酸铜组ADG、F/G及表观养分消化率具有明显的优势;血清铜含量随饲粮铜水平的提高而发生显著变化,而高铜组组问血清锌差异不显著;蛋氨酸组粪铜排泄量比硫酸铜组粪铜排泄量显著降低.说明:添加中等水平的有机蛋氨酸铜吸收利用率比高剂量硫酸铜高,且可减少粪铜、锌的排泄.     

猪肾脏等组织中盐酸莱克多巴胺的HPLC检测方法及其残留消除

本研究建立了猪肝脏、肾脏、肌肉和脂肪中盐酸莱克多巴胺的高效液相色谱检测方法。方法的检测限为1ng/g,定量限为2 ng/g,肝脏的平均回收率在73.4%～83.2%,变异系数在2.1%～6.6%;肾脏的平均回收率在73.1%～95.5%,变异系数在3.8%～4.0%;肌肉的平均回收率在80.7%～82.5%,变异系数在4.9%～9.1%;脂肪的平均回收率在73.3%～78.8%,变异系数在2.9%～6.7%。对60头健康猪以18 mg/kg的剂量混饲给药28d,停药饲喂14 d。在给药7、142、8 d和停药1、2、3、7、9、14 d分别屠宰6头猪,取各组织进行残留量测定。结果显示：肾脏中残留量最高,肝脏其次,残留量在停药期间较给药期间显著降低。其中肾脏和肝脏中的残留量分别在停药14 d、停药9 d后降至定量限以下;肌肉和脂肪中的残留量显著低于肾脏和肝脏,给药28 d时,残留基本低于定量限;停药1 d时均低至检测限以下。     

α-单月桂酸甘油酯对断奶仔猪生长性能、粪样微生物和血清免疫因子的影响

本试验旨在研究α-单月桂酸甘油酯(α-GML)对断奶仔猪生长性能、粪样微生物和血清免疫因子的影响。选取270头25日龄"杜×长×大"三元杂交断奶仔猪,按性别一致、体重[(6.74±0.86) kg]相近的原则分为3个组,每组6个重复,每个重复15头猪。对照组饲喂基础饲粮,试验组分别在基础饲粮中添加500和1 000 mg/kgα-GML。试验期21 d。结果表明:1)与对照组相比,饲粮添加1 000 mg/kgα-GML显著提高断奶仔猪平均日增重(P0.05),显著降低料重比(P0.05)。2)与对照组相比,饲粮添加1 000 mg/kgα-GML在断奶第14天及第21天时显著降低仔猪粪样中大肠杆菌和沙门氏菌的数量(P0.05),并在断奶第21天时显著增加仔猪粪样中乳酸菌的数量(P0.05)。3)与对照组相比,饲粮添加1 000 mg/kgα-GML在断奶第21天时显著降低仔猪血清免疫因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)及白细胞介素-1β(IL-1β)的含量(P0.05),对血清干扰素-β(IFN-β)的含量无显著影响(P0.05)。由此可见,α-单月桂酸甘油酯可显著提高断奶仔猪生长性能,减少肠道致病菌数目,缓解炎症。     

盐酸沃尼妙林预混剂在猪组织中残留消除规律研究

建立了猪肝脏、肾脏、肌肉和脂肪中盐酸沃尼妙林的高效液相色谱检测方法并研究盐酸沃尼妙林预混剂在猪体内各组织中的残留消除规律。对24头健康猪以200mg/kg的剂量混饲给药21d。在停药后0、6、12、18、24、36h分别宰杀4头猪,采集各组织进行药物残留测定。方法的检测限为0.025～0.062 5μg/g,定量限为0.05～0.1μg/g,肝脏的平均回收率为75.5%～76.4%,变异系数为2.3%～3.8%;肾脏的平均回收率为75.8%～78.5%,变异系数为4.1%～6.0%;肌肉的平均回收率为79.3%～80.0%,变异系数为3.0%～4.7%;脂肪的平均回收率为76.7%～77.3%,变异系数为3.3%～5.4%。结果表明,盐酸沃尼妙林在肝脏中残留量最高,肾脏其次;肌肉和脂肪中的残留量显著低于肝脏和肾脏,停药24h时,残留量低于定量限;停药36h时残留量均低至检测限以下。盐酸沃尼妙林预混剂在猪组织中消除迅速,建议休药期为2d。     

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid) poisoning in pigs

Young pigs, six to ten weeks of age, from two unrelated swine operations were fed a grower ration obtained from a common commercial supplier. Following ingestion of the feed for approximately two weeks, pigs in both groups developed neurological disturbances characterized by blindness, ataxia, incoordination, muscle tremors, posterior paralysis, and quadriplegia. Vocalization described as “screaming” was also observed in several animals. Necropsy findings and tissue arsenic concentrations were consistent with a diagnosis of phenylarsonic acid poisoning. The liver and kidney contained an average arsenic content of 2.9 and 1.8 mg/kg (wet weight), respectively. The feed contained 38 mg of arsenic/kg corresponding to 133 mg roxarsone (3-nitro-4-hydroxyphenylarsonic acid)/kg. This level of roxarsone is approximately three to five times higher than the levels recommended for swine rations. The feed company had placed roxarsone in the ration at levels recommended for the less toxic arsanilic acid. It was assumed that the two organic arsenicals could be added to the rations interchangeably at the same level of formulation. The present investigation indicated that roxarsone is more toxic than arsanilic acid and the margin of safety in swine rations is low.     

Technical note: a monoclonal antibody-based immunoassay for determination of ractopamine in swine feeds

An ELISA was developed for routine screening of ractopamine in swine feeds. Swine feed samples were extracted and purified, and the aqueous portion of the extract was analyzed for ractopamine using ELISA and confirmed by HPLC. For swine complex feeds containing ractopamine at 2.5 to 40 mg/kg, the average recoveries ranged from 73.1 to 86.5% by ELISA and 81.9 to 98.2% by HPLC. For the swine supplement containing ractopamine at 50 to 400 mg/kg, the average recoveries were 105.5 to 111.4% by ELISA and 89.1 to 92.9% by HPLC. The limit of detection was 0.24 microg/g by ELISA and 0.48 microg/g by HPLC, respectively. Results from the swine complex feeds (P = 0.009) and the supplement (P = 0.005) using ELISA and HPLC were not highly correlated. The ELISA was more sensitive and rapid and less expensive than the HPLC method and could be used for ractopamine screening in swine feeds before confirmation and quantification by other methods, such as HPLC.     

Toxic effects of supplemental copper and roxarsone when fed alone or in combination to young pigs

Three experiments were conducted to investigate the roxarsone (3-nitro-4-hydroxyphenylarsonic acid) X copper (Cu) interaction in weanling pigs. Supplemental roxarsone at 400 mg/kg diet decreased rate and efficiency of weight gain and caused visible neurological signs of toxicosis. Copper addition (CuSO4 X 5H2O) at a level of 650 mg Cu/kg diet likewise decreased weight gain and feed efficiency, and it also increased hepatic Cu deposition. The combination of these growth-depressing dosages of roxarsone and Cu resulted in a far greater reduction in gain and efficiency of feed utilization than was the case when either compound was fed alone. A growth-promoting dosage of Cu (250 mg/kg) increased weight gain by 32% in one experiment but showed no efficacy in alleviating the growth-depression resulting from feeding 400 mg/kg roxarsone. A roxarsone dosage of 100 mg/kg increased gain and feed efficiency. Surprisingly, the decreased weight gain in pigs fed 650 mg/kg Cu was ameliorated by feeding 100 mg/kg roxarsone concomitantly. This level of roxarsone also reduced liver Cu concentration substantially. It thus appears that the nature of the roxarsone X Cu interaction is dependent on the dose of each compound administered. Moreover, low-dose roxarsone administration appears to ameliorate Cu toxicity, but low-dose Cu feeding does not show efficacy against roxarsone toxicity.     

Arsenic-sulfur amino acid interactions in the chick

Experiments were conducted with growing crossbred chicks to determine the reasons why cysteine exacerbates roxarsone (3-nitro-4-hydroxyphenylarsonic acid) toxicity. A fortified corn-soybean meal diet that met or exceeded all nutrient requirements of the young chick was fed. While cysteine enhanced roxarsone toxicity, it had little effect on the toxicity of the inorganic arsenicals As2O3 and As2O5. The toxicity of another pentavalent organic arsenical, phenylarsonic acid, was also exacerbated by cysteine. In contrast, the growth-depression resulting from feeding the trivalent form of phenylarsonic acid, i.e., phenylarsine oxide, was not affected by dietary addition of cysteine. Supplementation of the diet with cystine, methionine or K2SO4 did not exacerbate roxarsone toxicity. Reduced glutathione (GSH), however, slightly increased the gain/feed depression resulting from feeding 300 mg roxarsone/kg diet. When injected ip 1) roxarsone and cysteine, or 2) roxarsone and ascorbic acid killed 100 or 60% of the birds, respectively, within 48 h postinjection. Few (6.7%) deaths resulted from ip injections of the same level of roxarsone alone. Therefore, the potentiation of toxicity requires pentavalent organic arsenicals and compounds that can act as reducing agents. We concluded that cysteine exacerbates roxarsone toxicity by reducing it to the more toxic trivalent state.     

三聚氰胺在中华鳖组织中的代谢动力学研究

应用液相色谱-串联质谱监测技术研究了三聚氰胺药饵多次给药后在中华鳖体内的药物代谢动力学。结果表明,中华鳖血液、肝脏、肌肉中三聚氰胺浓度分别在8、4、2h时达到高峰;最高峰时血液、肝脏、肌肉中药物质量浓度平均分别为16.98、16.79和12.05mg/kg。在血液、肝脏和肌肉组织中三聚氰胺浓度的t1/2（ke）分别为24.11、107.75和36.49h。三聚氰胺在血液、肝脏、肌肉组织中的代谢过程均符合一室模型,相应的动力学方程分别为：C血液=17.08×（e-0.0287t-e0.0558t）,C肌肉=11.64×（e-0.0190t-e2.1425t）,C肝脏=15.72×（e-0.0064t-e2.5558t）。说明药饵给药后三聚氰胺在中华鳖体内消除较慢。     

唑虫酰胺在柑橘及其土壤中残留规律及风险评估

目的 通过唑虫酰胺在柑橘及土壤中开展的两年三地的残留试验并进行风险评估,为制定唑虫酰胺在柑橘上的最大残留限量值提供数据支撑。方法 根据农药残留试验准则和标准操作规程开展田间试验,样品前处理后通过液相色谱-串联质谱测定得到试验数据,再进行风险评估。结果 唑虫酰胺在柑橘和土壤0.010~1.5 mg/kg的添加水平中,平均回收率达到90.6%~107.4%,相对标准偏差为1.5%~9.0%,定量限为0.010 mg/kg。两年三地唑虫酰胺在柑橘上的半衰期为17.3~34.7 d,土壤中的半衰期为5.8~34.7 d,末次施药后间隔21 d采集柑橘和土壤样品测定唑虫酰胺残留量均低于美国规定的最大残留限量值,不存在膳食摄入风险和急性膳食风险。结论 按照美国规定的最大残留限量值(MRL)1.5 mg/kg,间隔21 d采集的柑橘是相对安全的,唑虫酰胺的残留风险较低。     

Physiological and digestive effects of Neotyphodium coenophialum-infected tall fescue fed to lambs

The digestive responses and degradation of ergovaline and production of lysergic acid in the rumen of sheep offered Neotyphodium coenophialum-infected tall fescue straw at 2 ergovaline levels were investigated. Six crossbred wethers (56 +/- 3.0 kg of BW) were used in a randomized crossover design involving 2 treatments, for a total of 6 observations per treatment. The experiment consisted of two 28-d feeding periods with a 14-d washout period between them. The treatments were 1) tall fescue straw containing <0.010 mg of ergovaline/kg (E-), and 2) tall fescue straw containing 0.610 mg of ergovaline/kg (E+). Feed, orts, and feces were measured and analyzed for DM, ADF, and CP, and used to determine digestibilities. Feed and water intake were monitored throughout the feeding periods. Body weight and serum prolactin levels were measured at the beginning and end of each feeding period. Ruminal fluid was sampled 3 times (d 0, 3, and 28) during each 28-d feeding period for determination of ergovaline, lysergic acid, ammonia, and pH. Samples were collected before feeding (0 h) and at 6 and 12 h after feeding. Total fecal and urine collection commenced on d 21 and continued until d 25 of each feeding period. Ruminal ammonia, ruminal pH, and rectal temperature were not influenced by ergovaline concentration (P > 0.10). Digestion of DM, ADF, and CP was not different between treatments (P > 0.10). Daily water intake was less for the E+ diet (2.95 vs. 2.77 L/d; P < 0.05) as was serum prolactin (22.9 vs. 6.4 ng/mL; P < 0.05). Ergovaline concentration in ruminal fluid increased over sampling days at each sampling time (P < 0.05). Lysergic acid concentration in ruminal fluid increased over time from d 0 to 3 (P < 0.05) but was not different between d 3 and 28 (P > 0.10). In the E+ treatment, ergovaline was not detectable in the urine, whereas the concentration in the feces was 0.480 mg/kg. Lysergic acid was detected in the diet of the E+ treatment at 0.041 g/kg, lysergic acid in the urine was 0.067 mg/kg and in the feces was 0.102 mg/kg. The apparent digestibility of the alkaloids was 64.2% for ergovaline and -12.5% for lysergic acid. Approximately 35% of dietary ergovaline and 248% of dietary lysergic acid were recovered in the feces and urine. The appearance of lysergic acid in the feces, urine, and ruminal fluid is likely due to microbial degradation of ergovaline in the rumen and further breakdown in the lower digestive tract.     

饲料添加砷制剂对小鼠铁吸收的影响

为探讨饲料中添加砷制剂对小鼠铁吸收的影响,45只小鼠被随机分为3组,各组在基础日粮中分别添加0,40,80mg／kg砷（以洛克沙胂形式）,试验期30d。试验结束后,收取各组粪便和组织器官测定铁含量。结果显示,与对照组相比,40mg／kg砷组铁表观吸收率提高14．57％,80mg／kg砷组铁表观吸收率则降低了14．93％;40mg／kg砷组小鼠肝脏、肾脏、脾脏、小肠和腿肌铁含量显著升高（P〈O．05）,80mg／kg砷组则显著降低（P〈O．05）;但两组分别于停砷后5,7d,组织器官铁恢复正常水平。这表明40mg／kg砷水平可促进铁的吸收,相反80mg／kg砷水平则抑制铁的吸收。