A comparison of the test tube and the dialysis tubing in vitro methods for estimating the bioavailability of phosphorus in feed ingredients for swine

The validity of a simplified in vitro test tube (TT) method was compared with a more complicated dialysis tubing (DT) method to estimate the percentage of available phosphorus (P) in 41 plant origin and five animal origin feed ingredients for swine. The TT method using 1.0 or 0.25 g samples was compared with the DT method using 1.0 g samples at two pancreatic incubation times (2 vs 4 h) in a 3 x 2 factorial arrangement of treatments. Each DT and TT method treatment was replicated three and six times, respectively. Both methods utilize three enzymatic digestions: (i) predigestion with endoxylanase and beta-glucanase for 1 h, (ii) pepsin digestion for 2 h, and (iii) pancreatin digestion for 2 or 4 h. For the TT method, the entire procedure was conducted in a 50 mL conical centrifuge tube and replicated six times. For the DT method, the first two digestions were conducted in a 10 mL plastic syringe before the contents were quantitatively transferred into a segment of DT for the pancreatic digestion. The percentages of hydrolyzed P for plant origin ingredients measured by the DT method using 1.0 g samples and the TT method using 0.25 g samples were highly correlated (r = 0.94-0.97, P < 0.001) with each other and with published in vivo available P values for swine. Repeatabilities for these two methods ranged from 99.64 to 99.86%. The TT method using 1.0 g samples, however, did not provide valid estimates of P availability for all ingredients. For animal origin ingredients, neither method was significantly correlated (r = 0.1-0.6, P >or = 0.4) with published in vivo available P values. In conclusion, the accuracy and validity of the TT method using 0.25 g samples with a 2 h pancreatic digestion time was equal to or superior to the DT method using 1.0 g samples with a 4 h pancreatic digestion time for estimating P availability in plant origin feed ingredients.     

浙江省重点地区猪粪中重金属含量及安全施用评估

【目的】 浙江的规模养殖集中度高,畜禽排泄物资源化利用率达96%以上,通过堆肥和制作有机肥还田是主要的消纳途径,因此粪便中的重金属含量影响土壤安全及作物的生长。饲料是猪粪重金属最主要的来源,因此,本研究检测了浙江省猪饲料和排泄物中的重金属,为作物施用的安全性评价提供基础数据。 【方法】 采集35家不同规模养殖场的仔猪、生长猪和育肥猪3个阶段饲喂的饲料和粪便样本共566个,采用干灰化法、高压消解法消解,用AAS、AFS、ICP-MS法分析了铁、铜、锰、锌、砷、铅、铬、镉和汞9种重金属含量。 【结果】 1) 粪便中8种重金属平均含量趋势为Zn > Cu > Mn > As > Cr > Pb > Cd > Hg,主要残留元素为Zn、Cu、Mn和As,按干物质计其平均值分别为2405、1288、528和28.2 mg/kg,变异系数 ( CV) 分别为83%、34%、6%和15%。猪粪中Cr、Pb、Cd元素含量较低,在0.29～3.52 mg/kg之间,Hg含量仅3.97 μg/kg。猪粪中重金属含量仔猪阶段趋高,育肥猪阶段趋低,各个阶段变异都较大,变异系数CV值介于21%～112%之间,显示各地的生猪饲养中微量元素添加还比较混乱。2) 饲料有机砷的使用存在明显区域差异,萧山地区占72.7%的饲料样本在各阶段猪饲料中均添加有机砷,衢州地区的占33.3%,两个地区各有1家养殖场未使用有机砷。猪粪中54.9%样本砷含量 > 15 mg/kg,16.9%样本砷含量 > 50 mg/kg,2.8%的粪便砷超过70 mg/kg。3) 按畜禽粪便安全使用准则进行判定,69.9%猪粪铜超标,16.9%猪粪砷超标。按有机肥料标准进行判定,猪粪中54.9%样本砷含量超标。按照饲料中金属元素在粪便中的浓缩率:Fe、Zn、Cu为7～10倍,锰约为3倍,当前饲养水平下,Fe、Zn、Cu元素存在过量添加。4) 以浙江全省旱地单位面积承载量估算,当前能消纳猪粪中Zn、Cu的排放,但当每公顷猪粪施用量超过2.27 t (干计) 时,局部地区重金属就有累积风险。 【结论】 应降低猪饲料中微量元素的添加量,禁止饲用有机砷制剂,才能有效降低猪粪施用所导致的重金属污染;按照目前监测猪粪肥重金属元素含量水平,有机肥的施用量用应控制在2.27 t/hm2 (干基) 以内,以防止对土壤造成污染。      

Improved turbidimetric assay of tylosin in premix and animal feeds not containing urea

A turbidimetric method is described for the determination of tylosin in premix and animal feeds not containing urea. This method includes several modifications of existing tylosin turbidimetric and AOAC plate assays to remove interferences from the feed, to concentrate low levels of tylosin, and to reduce the variability of the assay results. An acidic alumina column cleanup step has been incorporated into the method to remove interferences from feed ingredients. A disposable C18 column was used to concentrate the tylosin from low-level feeds, and the use of a larger analytical sample size has decreased the variability of the assay results. Average recoveries of tylosin added to chicken and swine rations were 98 and 101%, respectively.     

Liquid chromatographic determination of oxfendazole in swine feeds

A relatively simple analytical method is presented for determination of oxfendazole (2-(methoxycarbonylamino)-5-phenylsulfinyl-benzimidazole) at levels as low as 0.012% in swine feeds, using cation exchange liquid chromatography (LC). The sample was extracted with a solvent mixture of methanol-glacial acetic acid (90 + 10) at 45 degrees C, using a gyrorotory shaker. Plant pigments and other feed excipients were removed using zinc acetate treatment and pH-controlled extraction. Oxfendazole was further separated from the remaining interferences and quantitatively determined by LC on a Partisil SCX column with acetonitrile-0.01M phosphate buffer as mobile phase. The method is stability-specific, linear, precise, and accurate at 80-120% labeled strength (relative standard deviation 0.9-1.7 with mean recovery of 98-99%). Supporting data at a level of 0.0135% oxfendazole in swine feed indicated that this method is capable of complete recovery of oxfendazole from medicated swine feeds.     

基于智能手机平台的猪饲料配方系统设计与实现

设计依猪只个体日龄、体重等参数而异的饲料配方是实现猪精细饲养的关键，目前已有很多具备此功能的饲料配方软件，但它们大多数基于PC平台。结合智能手机便于携带、可移动性好等优点开发的基于智能手机平台的猪饲料配方软件，便于携带且操作简单，使用NRC1998模型精确预测了猪营养需要量，配方结果更准确科学，饲料原料营养成分数据用XML文件贮存，数据读取方便，有效地降低了系统开发成本。     

Contact between root and iron substrate and the mobilization of iron by soybean roots

The question of whether a direct (uninterrupted) contact between plant roots and solid substrate is a prerequisite for the mobilization of sparingly soluble iron (Fe) by plant roots was investigated. Factorial combinations of two types of contact between the roots and rooting medium [direct or interrupted (roots placed inside a dialysis tube (DT)], two rooting media (nutrient solution or vermiculite), and two Fe supply conditions [without soluble Fe (control) or plus Fe‐EDDHA] were investigated. Placing the roots inside DT, with no visible mechanical impedance for the root, reduced the growth of tops and roots in most cases. Iron mobilization, judged by the total amount of Fe in the tops, strongly decreased if roots were enclosed in DT. This was true in plants grown in vermiculite as well as in nutrient solution containing Fe‐EDDHA. It is concluded that a direct contact between the surface of root cell walls and the Fe substrate is a prerequisite for the Fe mobilization by the plant roots.     

Rapid automated turbidimetric assay for chlortetracycline hydrochloride, using Leuconostoc mesenteroides as the test organism.

A rapid turbidimetric assay has been developed for chlortetracycline hydrochloride (CTC-HCl) in finished animal feeds and feed supplements, using Leuconostoc mesenteroides as the test organism. Two modifications are presented: The incubation period of modification 1 is 2.5 hr and the sensitivity of the assay is 0.03 microgram CTC-HCl/assay tube. Modification 2 has a sensitivty of 0.01 microgram CTC-HCl/assay tube and requires an incubation period of 3.5 hr. For 21 feed formulations, the turbidimetric method recovered 95.7% of label claim. Recoveries of CTC-HCl standards from the same feeds ranged from 93.4 to 134% with a mean of 103%. The relative standard deviation among day-to-day duplicates is 3.50% for the faster modification and 1.63% for the more sensitive modification.     

Microbiological plate and turbidimetric assays of chlortetracycline in feeds: collaborative study.

The manual and automated turbidimetric assays and a modified official plate assay for chlortetracycline (CTC-HCl) in feed were collaboratively studied. Three feed samples (swine feed, 100 g CTC-HCl/ton; premix I, 20 g each of CTC-HCl and sulfamethazine/lb, and 10 g penicillin/lb; and premix II, 50 g CTC-HCl/lb) were analyzed at 2 dilutions. Twelve laboratories conducted the plate assay; 8 laboratories the manual turbidimetric method; and 7 laboratories, the Autoturb analysis. Within a method, there was no significant difference between dilutions. Between methods, there was a significant difference between the manual turbidimetric plate assays only for swine feed. However, the same sample dilutions or the average values of the 2 dilutions for both methods showed no statistical difference. Among the collaborators, the slope of CTC-HCl standard curve varied between about 2.0 and 3.0 for the plate method. The turbidimetric assay has been adopted as official first action for feeds containing larger than or equal to 20 g CTC-HCl/lb.     

矿山生态型水蓼对施用猪粪土壤中磷的吸取净化效果

【目的】 针对猪粪不合理利用导致土壤中磷过剩和磷流失的突出问题,明确磷富集植物矿山生态型水蓼 (Polygonum hydropiper) 对施用猪粪土壤磷的吸取净化效果,为高效提取施用猪粪土壤中的过量磷,降低磷素的流失风险提供科学依据。 【方法】 采用微区模拟试验,根据农田磷肥安全用量 (< 200 kg/hm2),设1、2、3 kg/m2共3个猪粪用量,以不施猪粪为对照 (CK),共4个处理,每处理3次重复。在矿山生态型水蓼收获期 (移栽后3个月) 采集植物地上部,采用微波消解仪 (CEM MARS5, USA) 消解—全自动间断化学分析仪 (AQ2, UK) 测定植株磷含量,分析矿山生态型水蓼对猪粪处理土壤中磷的富集能力。在矿山生态型水蓼移栽前和收获后按5点采样法分别采取0—20 cm和20—40 cm土层土壤样品,测定土壤样品中的水溶性磷和有效磷含量,分析种植矿山生态型水蓼前后猪粪处理土壤中易溶性磷含量的变化情况。 【结果】 不同用量猪粪处理下,矿山生态型水蓼地上部生物量较不施猪粪处理分别显著增加了18.4、24.6和42.0 g/株,最大时高达不施猪粪处理的2.16倍。各猪粪处理矿山生态型水蓼地上部磷含量显著高于不施猪粪处理,较不施猪粪处理分别增高了0.60、0.91和1.49 g/kg,最高为施猪粪处理的1.53倍。矿山生态型水蓼地上部磷积累量随猪粪用量的增加而显著增大,较不施猪粪处理分别增加了P 84.0、124和236 mg/株,最高为不施猪粪处理的3.32倍。各处理下,种植矿山生态型水蓼后0—20 cm土层中的水溶性磷和有效磷含量与种植前相比均显著降低,水溶性磷含量降低了74.9%～81.5%,有效磷含量降低了48.9%～60.0%;20—40 cm土层的水溶性磷和有效磷含量均无明显变化。 【结论】 种植矿山生态型水蓼能显著减少表层土壤中水溶性磷和有效磷量,高效吸取净化猪粪带入土壤中磷。因此,在高磷含量的土壤上种植、收获矿山生态型水蓼,是降低土壤磷素流失风险的有效生物手段。      

Determination of virginiamycin in feeds.

Virginiamycin was extracted from the feed by ethanol-pH 2.5 phosphate buffer (1 + 1). The pH during extraction was adjusted (when necessary) to between 4 and 5. Sample dilutions and the standard dose response line were prepared to contain ethanol pH 6 phosphate buffer (2 + 8), and the test organism was Sarcina lutea. Three feeds (a poultry ration, a swine finishing ration, and a swine starter ration) showed virginiamycin recovery of 88.8--108.9% when standard solutions were added at concentrations of 4.54--90.8 g/ton. The coefficient of variation (4--20%) was larger for low potency feeds (10 g/ton) compared to the higher feeds (100 g/ton). Similarly, excellent recovery was obtained when the swine starter feed was fortified by a commercial premix. Amprolium, roxarsone, and monensin can be present at 20 times the concentration of virginiamycin with little or no interference in the antibiotic determination. Lasalocid at 10 times the concentration of virginiamycin caused a slightly positive bias (recovery, 107.4%).     

Identification, characterization, epitope mapping, and three-dimensional modeling of the alpha-subunit of beta-conglycinin of soybean, a potential allergen for young pigs

Soybean meal (SBM), the major byproduct of soybean oil extraction, is the main protein source for swine diets globally. In the United States, 8.6 million metric tons of SBM was used in swine rations in 2004. The pathological effect and immunological response of SBM feeding have been demonstrated in swine. In this study, we have utilized plasma collected from piglet feed with SBM in immunoblot analysis to detect proteins that elicited antigenic responses. We have identified soybean beta-conglycinin alpha-subunit as being a potential allergen for young piglets. Characterization of this protein indicated that deglycosylation and pepsin digestion did not eliminate immunoreactivity of this protein. Epitope mapping utilizing planar cellulose supports technology (SPOT) showed that three peptides spanning amino acids S185-R231 were critical for the allergenicity. A computer-generated three-dimensional structure model of the alpha-subunit of beta-conglycinin indicated that the antigenic epitopes were located on the surface of the protein. Information from this study may assist in the construction of recombinant nonallergenic soybean protein useable for both immunotherapy and the potential production of hypoallergenic soybean plants.     

Gas chromatographic determination of incurred dimetridazole residues in swine tissues

A gas chromatographic method for determination of 2-hydroxymethyl-1-methyl-5-nitroimidazole (DMZOH), the hydroxy metabolite of dimetridazole, in swime muscle has been developed. The method uses cleanup steps similar to those of an earlier polarographic method. The present method is capable of quantitating levels as low as 2 ppb and detecting less than 1 ppb. Recoveries from 30 control tissues spiked at 1, 2, or 4 ppb averaged 80.4%. Performance of the method in incurred tissue was documented and limited data on the depletion of the metabolite in muscle were generated. The muscle of swine given 150 ppm dimetridazole in feed for 14 days contained less than 1 ppb DMZOH at 12 h withdrawal time.     

A rapid dichromate procedure for routine estimation of total nitrogen in soils

Abstract

A simple procedure for routine estimation of total nitrogen (N) in soils is described. It involves (a) digestion of the soil sample with an acidified dichromate (K₂Cr₂O₇‐H2SO₄) solution for 45 minutes in a Pyrex digestion tube in a 40‐tube block digester preheated to 170°C, (b) titrimetric determination of the ammonia‐N liberated by direct steam distillation of the digest with alkali (NaOH), and (c) calculation of the total‐N value by application of a correction factor (1.1) to the measured NH₃‐N value. Studies using 14 soils selected to obtain a wide range in total N content and other properties showed that the uncorrected recovery of N by the procedure described ranged from 88.5% to 92.3% and averaged 90.5%. When a correction factor of 1.1 was applied to the results, the recovery of N ranged from 97.4% to 101.5% and averaged 99.6%.

The dichromate procedure described is rapid and precise, and it permits at least 80 analyses in a normal working day. The only equipment required for its use is equipment commonly used for total N analysis of soils by the Kjeldahl method (40‐tube block digester and steam distillation unit), and the acidified dichromate solution used for digestion of the soil is, the reagent commonly used for determination of organic carbon in soil by a modified Mebius procedure. Besides being simpler and more rapid than the Kjeldahl method, the procedure described has the advantage that it does not require the expensive fume disposal system needed for Kjeldahl digestion.     

Detection of low-level carbadox residues in animal feeds by high pressure liquid chromatography

The high pressure liquid chromatographic (HPLC) method is capable of detecting from 1 to 0.024 ppm methyl 3-(2-quinoxalinyl-methylene)carbazate-N1,N4-dioxide (carbadox). Carbadox is extracted from the feed with 2% NH4OH in acetone, passed through a liquid-liquid partition, subjected to HPLC, and detected by using a 365 nm detector. No feed materials or other active drug ingredients produced false positive results.     

Microbiological method for assaying lincomycin in animal feed: collaborative study.

A microbiological assay for determining lincomycin in swine feed, supplement, and a vitamin-mineral premix was studied collaboratively in 16 laboratories. The design of the study involved a complete feed, feed supplement, and a vitamin-mineral premix covering a range of fortification from 20 to 80 g/ton and 80 to 2600 g/ton. Two methods of sample preparation were used depending on the concentration of lincomycin in the sample. Statistical evaluation of the results from the 2 methods indicated that 10 and 11 collaborators, respectively, had mean recoveries which were not significantly different from one another. Ten laboratories obtained a mean recovery of 112.2% (range 102.3--123.5%) for the lower level, and 11 laboratories obtained a mean recovery of 104.4% (range 100.0--107.7%) for the higher level. The method has been adopted as official first action.     

Residue depletion of ractopamine and its metabolites in swine tissues, urine, and serum

Ractopamine hydrochloride is a beta-adrenergic leanness-enhancing agent approved for use in swine in the United States. Depletion of ractopamine and its metabolites from animal tissues, urine, and serum is of interest for the detection of illegal use. The objectives of this study were to measure the residues of ractopamine in swine incurred samples after treatment with dietary ractopamine for 28 consecutive days. An efficient and sensitive analytical method was developed for the detection of parent ractopamine and its metabolites in swine tissues, urine, and serum by HPLC-FLD. After extraction, enzymatic digestion, and solid-phase cleanup of the samples, ractopamine residues were determined by liquid chromatography (LC) with fluorescence detector. The limits of detection (LOD) for tissues, urine, and serum were 1 ng g(-1), 0.5 ng mL(-1), and 0.5 ng mL(-1), respectively. Recoveries ranged from 70.5 to 94.5% for samples fortified at 1-50 ng g(-1) or ng mL(-1). Sixty pigs were fed twice daily for 28 consecutive days with feeds containing 18 mg kg(-1) ractopamine HCl. The residue concentrations in urine, liver, and kidney were 650.06 ng mL(-1), 46.09 ng g(-1), and 169.27 ng g(-1), respectively, compared with those in muscle, fat, and serum (4.94 ng g(-1), 3.28 ng g(-1), and 7.48 ng mL(-1), respectively) at the feeding period of 7 days. The residue concentrations at withdrawal period of 0 days in all edible tissues were lower than tolerance values established by the FDA and MRL values listed by the JECFA. These data support the withdrawal time of 0 days established by the FDA for ractopamine used as feed additive in swine.     

A rapid and precise method for routine determination of organic carbon in soil

Abstract

A simple method for routine determination of organic carbon in soil by a modified Mebius procedure is described. It involves (a) digestion of the soil sample with an acidified dichromate (K₂Cr₂O₇‐H₂SO₄) solution for 30 minutes in a Pyrex digestion tube in a 40‐tube block digester preheated to 170°C and (b) estimation of the unreacted dichromate by titration of the cooled digest with an acidified solution of ferrous ammonium sulfate with use ofN‐phenylanthranilic acid as an indicator. The method is more rapid and precise than the Mebius procedure commonly used for routine analysis of soils for organic carbon, and the only equipment required for its use is equipment now commonly used for routine Kjeldahl analysis of soils for total nitrogen.     

Analysis of patulin in apple juice by diphasic dialysis extraction with in situ acylation and mass spectrometric determination.

A procedure combining diphasic dialysis extraction with in situ acylation and gas chromatography/mass spectrometry (GC/MS) determination was developed for detection and quantification of the mycotoxin patulin in apple juice. Apple juice samples spiked with 4-N,N-dimethylaminopyridine were dialyzed using methane chloride and acetic anhydride inside dialysis tubing. Patulin was derivatized into its acetate and collected in the tubing after diphasic dialysis and was directly determined using GC/MS with the selective ion monitoring mode without further concentration and cleanup steps. Quantification was carried out by a calibration curve with an internal standard of correlation. The appropriate parameters of both dialysis and derivatization were examined. The linear range of the calibration curve was found to be 10-250 microg/L for patulin, and the limit of quantification was 10 microg/L. Levels of patulin ranging from 0 to 107.2 microg/L with 77-109% recovery were found in 10 apple samples. The technique combining diphasic dialysis extraction and acylation was demonstrated and showed potential for other applications.     

In vitro dissolution and in vivo absorption of calcium [1-(14)c]butyrate in free or protected forms

Butyrate is a byproduct of microbial carbohydrate fermentation that occurs primarily in the large intestine. When added to feed, butyrate quickly disappears in the upper digestive tract. Because butyrate is important for epithelial cell development, mucosal integrity, and animal growth, an encapsulation technique has been developed that allows for the slow release of butyrate into the small and large intestines. The purpose of this study was to describe the in vitro release of calcium [1-(14)C]butyrate, formulated into a slow-release (protected) bead, into water and simulated intestinal fluids and to compare the in vivo absorption and disposition of unprotected versus protected calcium [1-(14)C]butyrate in broiler chicks. Formulation of calcium [1-(14)C]butyrate into protected beads allowed release of 5.8 ± 0.2 and 3.4 ± 0.2% of the formulated radiocarbon into water and gastric fluid, respectively, after 2 h of incubation. Beads incubated in gastric fluid for 2 h and subsequently incubated in simulated intestinal fluid released a total of 17.4 ± 0.8% of the formulated radioactivity. Release of respiratory [(14)C]CO(2) after oral dosing of aqueous calcium [1-(14)C]butyrate in broiler chicks peaked at 15.2 ± 5.2% per hour 1.5 h after dosing; in contrast, maximal rates of release in chicks dosed with protected calcium [1-(14)C]butyrate occurred 4 h after dosing at 9.0 ± 3.1% per hour. The data suggested an improved efficacy of protected butyrate delivery to intestinal tissues over nonprotected butyrate. This study confirmed that encapsulation strategies designed to enhance delivery of ingredients to improve intestinal health are effective at prolonging intestinal exposure to butyrate. Encapsulation of such ingredients might benefit the food and feed industries.     

Comparison of four methods of digestion for the determination of total phosphorus content in hen droppings

Abstract

The objective of this study was to compare four methods of digesting for the determination of total phosphorus (P) content in hen droppings by colorimetric spectrometry. For this comparison, we used an homogeneous and oven‐dried sample of hen droppings and made 19 analyses with each method on this same sample. Method 1 was a dry and complete digestion with addition of calcium carbonate before ashing at 550°C for 12 h. Method 2 was a dry simplified digestion without the addition of calcium carbonate. Method 3 was a wet and complete digestion with sulfuric acid and successive addition of nitric acid and heating. Method 4 was a wet simplified digestion with sulfuric and nitric acids added at once and heating for 30 min. We obtained P levels of 1.93±0.049%, 1.93±0.047%, 1.96±0.079%, and 1.98±0.143% for the four methods, respectively. There was no statistical difference among those four methods, and therefore we recommend Method 2 since it requires the use of less concentrated acids, and in addition, the standard deviation is the lowest.