Identification of species-specific DNA in feedstuffs

Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.     

生物素缺乏及肉鸡和种鸡日粮中添加生物素效果的研究

５４０只１日龄红布罗商品代雏鸡随机分为９组，第１组饲喂以小麦为基础的诱发日粮，第２和第３组在１组基础上分别添加３００（μｇ／ｋｇ日粮）生物素和动物脂肪（前期２％，后期３％）。第４至９组饲以玉米－豆饼型日粮，前后期分别添加０，５０，９０，１２０，１５０，２００和０，５０，７０，９５，１２０，１６０（μｇ／ｋｇ日粮）的生物素。结果表明：生物素缺乏造成足底皮肤炎，肝脏脂肪酸成分发生变化：不饱和脂肪酸（棕榈油酸）显著增加（Ｐ＜０．０１），饱和脂肪酸（硬脂酸）减少（Ｐ＜０．０５）。２９日龄绝食２４ｈ，饲喂诱发日粮的鸡，血糖、肝糖原和肝脏总脂肪含量下降，血浆甘油三酯增加，但无显著差异（Ｐ＞０．０５）。玉米－豆饼型日粮中添加不同水平生物素，绝食后血糖、肝糖原和甘油三酯含量未发生显著变化，随生物素添加水平提高，对足部皮肤状况有一定的改善作用，前期添加１５０或２００（μｇ／ｋｇ日粮）生物素对增重和饲料报酬较好，而后期添加生物素影响不大。种鸡试验选用３３６只２０周龄育成种鸡随机分为３组，分别饲以添加有０，１００，１５０μｇ／ｋｇ生物素的日粮。初步结果表明：生物素对蛋重无影响，日粮添加１００和１５０μｇ／ｋｇ生物素可提高受精率０     

Early weaning of calves using feedstuffs. A rationalization based on inhibition of lipolysis

The ability of broll (a combination of the wheat-milling byproducts bran and pollard, i.e., a mixture of wheat bran, husk, and flour) and blackstrap molasses (an ingredient of calf feed) to inhibit calf pregastric lipase (CPGL)-catalyzed hydrolysis of tributyrylglycerol (TBG) has been studied in vitro. Lipolysis was measured at pH 6.5 and 37 degrees C (CPGL at 0.02 mg/mL) with stirring at 300 rpm. The broll soaked in Bis-Tris buffer (50 mM, pH 6.5) at 4 degrees C for either 24 h or 15 min, and then added to an emulsion containing TBG, before initiation of the reaction by addition of CPGL, exhibited 22% inhibitory effect. A solution of blackstrap molasses (50%, v/v) exhibited inhibitory effects of 50% in the absence and 45% in the presence of Bis-Tris buffer. The initial rate of lipolysis in the presence of the dialyzed molasses retentate (10%, v/v) increased a little, compared with the same amount of crude molasses, from a mean value of 69% to a mean value of 74%. The results have been discussed in terms of the chemical nature and composition of broll and molasses and their roles as components of feedstuffs used in development of the rumen in early weaning of calves.     

Phytase and acid phosphatase activities in plant feedstuffs

A total of 183 samples representing 24 feedstuffs were analyzed for total phosphorus, phytate phosphorus content, phytase (Phy), and acid phosphatase (AcPh) activities with the objective to predict the capacity to hydrolyze phytic acid and to contribute to formulating environmentally adequate diets for monogastric animals. Of the cereals and cereal byproducts analyzed, only rye (5147 U kg(-)(1); 21 955 U g(-)(1)), wheat (1637 U kg(-)(1); 10 252 U g(-)(1)), rye bran (7339 U kg(-)(1); 56 722 U g(-)(1)), and wheat bran (4624 U kg(-)(1); 14 106 U g(-)(1)) were rich in Phy and AcPh activities. Legume seeds and oilseeds contained negligible Phy activity and a moderate amount of AcPh activity, except for kidney bean (33 433 U g(-)(1)) and full-fat linseed meal (13 263 U g(-)(1)). On the other hand, a significant linear regression between phytate phosphorus (y) and total phosphorus (x) was observed in cereal byproducts (R(2) = 0. 95; y = 0.8458x - 0.0367; P < 0.001) and oil seeds (R(2) = 0.95; y = 0.945x - 0.20; P < 0.001). Phy and AcPh were positively correlated with respect to phytate phosphorus in cereals, cereal byproducts, and other byproducts and negatively correlated in legume seeds and oilseeds. Except for cereals, the highest correlation between enzyme activities and phytate phosphorus was found for phytase. It is not possible to predict Phy and AcPh activities from phytate phosphorus content by linear and quadratic regressions. Finally, only highly significant and positive correlation was found between Phy and AcPh activities for cereals, cereal byproducts, and oilseeds.     

Disposable cartridge extraction of retinol and alpha-tocopherol from feedstuffs

Automated determination of fat-soluble vitamins by modern methods such as liquid chromatography is hampered by the initial extraction step. A simple technique is proposed that allows an appreciable increase in the actual rates of determination. Feedstuff samples are first hydrolyzed in an aqueous alcohol (mainly methanol)-potassium hydroxide solution. Instead of extracting retinol and alpha-tocopherol from the hydrolysis solution by an organic solvent, an aliquot of the solution is mixed with a small volume of a strong antioxidant solution (ascorbic acid) and pipetted onto a kieselguhr disposable cartridge where it is adsorbed. Retinol and alpha-tocopherol are eluted with isooctane at normal pressure. The proposed method has been compared with conventional techniques on many feed samples.     

饲料原料种类在线识别系统设计与试验

入仓原料种类识别是饲料生产过程中的关键环节之一。目前,入仓原料主要通过人工取样的方式,依靠工人感官经验识别原料种类,以确保原料正确入仓。为了实现饲料原料种类在线自动取样和识别,提高饲料加工的自动化水平,该研究设计了一种多通道饲料原料自动取样装置,应用机器视觉技术,搭建了原料种类在线识别系统。该系统主要由取样单元、样品输送单元、图像采集单元等组成;采用Arduino Uno为系统控制核心,设计了控制流程和控制线路;在Arduino IDE开发环境下编写了控制程序;运用卷积神经网络的方法构建了饲料原料种类识别模型CAM-ResNet18;基于PyQt5环境开发了饲料原料种类在线识别系统软件,包括上位机人机交互软件系统和下位机自控控制系统。上位机系统软件通过串口与下位机控制器通讯,实现对饲料原料种类在线取样识别装置的自动控制。通过模型嵌入和系统集成,对系统的基本功能、识别准确率和识别时间进行测试。饲料原料种类在线识别系统运行正常可靠,实现了饲料原料入仓过程中的自动取样、图像采集、种类识别、结果反馈、一键报警的全环节智能操作。系统性能测试中,饲料原料种类识别准确率为98%,取样识别周期为10.13 s。研究结果表明开发的饲料原料种类在线识别系统可以实现入仓饲料原料在线取样和种类识别功能,为饲料加工中饲料原料种类的自动识别提供了新的方法和技术支撑。     

Detection of twelve mycotoxins in mixed animal feedstuffs, using a novel membrane cleanup procedure.

A multimycotoxin thin layer chromatographic screening method is described which is applicable to most animal feedstuffs. Interference from nonspecific lipid, pigment, and other components of simple and mixed feeds is reduced to a minimum by using a membrane cleanup step. Aflatoxins B1, B2, G1, and G2, citrinin, diacetoxyscirpenol, ochratoxin A, patulin, penitrem A, sterigmatocystin, T-2 toxin, and zearalenone may be reliably detected. The sensitivity of the method is generally low for mixed feeds but even so aflatoxin B1 can be detected at a level of 3 ppb and ochratoxin A at 80 ppb. While the basic method is less sensitive for sterigmatocystin (330 ppb), patulin (600 ppb), zearalenone (1000 ppb), and the trichothecenes (1000-4000 ppb), it may be adapted so as to reduce the above detection limits when the presence of these toxins is suspected. Lower levels may be detected in extracts of simple feeds.     

Fermentation of cottonseed and other feedstuffs in cattle rumen fluid

Bovine rumen fluid was fermented anaerobically over 48 h with cottonseed, corn, alfalfa, or a mixture of these substrates in anaerobic mineral buffer. Samples taken at different incubation times were derivatized with n-butanol and subjected to gas chromatography and mass spectroscopy. No unusual fermentation end-products from the cottonseed substrate were detected. Cottonseed supported rumen fermentation at levels comparable to those of the other substrates. Major components were usually found in the decreasing order of acetate, propionate, butyrate, and valerate, although acetate and propionate concentrations decreased late in the alfalfa and mixed-feed fermentations, eventually allowing butyrate concentrations to exceed those of propionate. As expected, lactate was produced in high concentrations when corn was fermented. The minor components 2-methylpropionate, 2- and 3-methylbutyrate, phenylacetate, phenylpropionate, and caproate also accumulated, with their relative concentrations varying with the substrate. Succinate was produced in substantial amounts only when corn and alfalfa were fermented; it did not accumulate when cottonseed was the substrate. Samples containing cottonseed were derivatized and subjected to reversed-phase high-performance liquid chromatography, revealing that gossypol concentrations did not change during fermentation.     

Liquid chromatographic determination of aflatoxins in feedstuffs containing citrus pulp

A liquid chromatographic (LC) method was developed for the determination of aflatoxins in feedstuffs containing citrus pulp. The feed-stuff sample is extracted with chloroform, followed by Sep-Pak Florisil cartridge cleanup and Sep-Pak C18 cartridge cleanup. The final eluate (water-acetone, 85 + 15, v/v) is submitted to reverse-phase liquid chromatography with water-methanol-acetonitrile (130 + 70 + 40, v/v/v) as mobile phase and postcolumn derivatization with iodine. Citrus components are removed from the extract efficiently. The limit of detection for aflatoxin B1 is less than 1 microgram/kg. Other aflatoxins can also be detected and measured. Recoveries of aflatoxins B1, B2, G1, and G2 for dairy rations spiked at 13, 5, 10, and 4 micrograms/kg were 87, 86, 81, and 82%, respectively. Corresponding coefficients of variation were 3.1, 3.6, 5.2, and 3.8%, respectively.     

Modified flotation technique for quantitative determination of mite populations in feedstuffs

Modifications are described to a previously published technique for quantitative determination of mite populations in animal feedingstuffs. In the modified method, the oil phase is kerosene instead of a mixture of kerosene and tetralin (1 + 1), and aqueous industrial methylated spirit is substituted for aqueous ethanol in the aqueous phase. Emulsion formation at the interface is considerably reduced by incorporating a pre-extraction stage. Examination of mites on the filter paper is made easier and quicker by staining them with Phloxin B. Trials carried out with known numbers of mites in samples of dairy feed concentrates indicate mean recoveries of 84-96% throughout the range tested. The results show that the accuracy of the technique is not affected by the level of infestation.     

Analyzing sulfur amino acids in selected feedstuffs using least-squares nonlinear regression

Five feedstuffs were oxidized using performic acid, and these, along with their unoxidized counterparts, were acid hydrolyzed for multiple times (0-144 h) in degassed and vacuum-sealed glass tubes. The methionine sulfone, cysteic acid, methionine, and cysteine contents were determined for each hydrolysis time. Least-squares nonlinear regression of the sulfur amino acid contents and hydrolysis time was used to predict the actual sulfur amino acid content as well as the hydrolysis and loss rates. Least-squares nonlinear regression estimates for methionine content compared well with those of methionine sulfone for most of the feedstuffs tested. In contrast, the estimates for cysteine agreed poorly with cysteic acid. The loss rates during acid hydrolysis for methionine, methionine sulfone, and cysteic acid were low. Overall, acid hydrolysis in an evacuated sealed tube for 24 h without prior oxidation is suitable for methionine, but not cysteine, quantitation in some complex feedstuffs.     

Rapid, economical method for determination of aflatoxin and ochratoxin in animal feedstuffs

A quantitative procedure widely used in European Economic Community (EEC) countries has been successfully scaled down to produce a rapid method for determination of aflatoxin B1 (and other aflatoxins) in animal feeds. Without modification, the method may be used for simultaneous ochratoxin A determination in simple feeds, but a slightly different extraction procedure is required for compound feeds. Validity of the method has been demonstrated by comparison with the full EEC procedure for aflatoxin B1 and the Nesheim method for ochratoxin A. Analyses may be completed within 2 h and there is a considerable savings in materials over the 2 reference methods. The procedure is also less hazardous because volumes of toxic extract are small, and the operator is exposed to minimum solvent vapor.     

温度和粉碎粒度对不同能量饲料原料热物理特性的影响

为探究常见能量饲料原料在不同温度、不同粉碎粒度下的热物理特性差异,该文以4种谷物(玉米、小麦、大麦和高粱)和4种加工副产品(小麦麸、木薯渣、甜菜渣和米糠)原料为研究对象,分别粉碎过孔径1.5、2.0和2.5 mm筛片,得到3种不同粉碎粒度的粉料,利用差示扫描量热法(differential scanning calorimetry,DSC)和线热源法分别测定了不同粉料在25～100℃范围内的比热和导热率,通过计算得到相应的导温系数,分析了温度、粉碎粒度对原料热特性的影响以及不同原料之间的热特性差异,并建立了热特性参数关于温度的回归预测模型。结果显示:8种原料粉碎过1.5 mm筛孔的粉料比热、导热率和导温系数随温度的升高分别在1.580～2.671 kJ/(kg·K),0.054～0.362 W/(m·K)和6.694×10~(-8)～23.254×10~(-8) m~2/s范围内变化。整体上,原料的热特性均随温度的升高而呈线性或非线性上升趋势。不同粉碎粒度的同一原料,在相同温度下的比热差异均不显著(P0.05);随着粒度的增大,粉料的导热率和导温系数均有逐渐下降的趋势。4种谷物的比热与温度之间呈线性关系;小麦麸、木薯渣和米糠的比热可用温度的三次多项式表示;而甜菜渣的比热与温度呈二次关系。4种谷物和甜菜渣在3种粒度下的导热率均可用温度的三次多项式表示,而小麦麸、木薯渣和米糠的导热率则可用温度的二次多项式表示。小麦麸和米糠的导温系数与温度呈二次关系,其余6种原料的导温系数则可用温度的三次多项式表示。研究结果可为配合颗粒饲料配方变换所需的调质、制粒等热加工过程的工艺参数的调整、优化提供理论依据。     

Occurrence of mycotoxins in cereals and animal feedstuffs in Natal, South Africa

During the period 1982-1983, just under 800 samples of agricultural commodities, comprising cereals, compound feeds, hay, and silage, were examined for molds and mycotoxins. Aflatoxin B1 showed the highest incidence rate; it occurred in over 27% of all samples analyzed, the highest levels being found in peanut meal at 1500 ppb. Other mycotoxins detected were patulin and a number of trichothecene toxins at incidence rates in all commodities of 5.6 and 3.1%, respectively. The commodities at highest risk were oil seeds, excluding soya bean; the latter was found to be fairly free from contamination with mycotoxins. The most prevalent fungi were Aspergillus flavus and parasiticus, which were found in over 22% of all samples, whereas Penicillium spp. showed the lowest incidence of genera, specifically identified in 8.3% of all samples examined. This latter finding explains in part the low incidence of Penicillium mycotoxins.     

Uptake and transfer of PCDD/Fs by cattle fed naturally contaminated feedstuffs and feed contaminated as a result of sewage sludge application. 2. Nonlactating cows.

The dietary absorption and tissue distribution of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) was investigated in 4 nonlactating Simmental cows. During Phase 1 the dietary uptake and fecal excretion of these chemicals were measured over 10 days using feed containing background levels of PCDD/Fs that were primarily of atmospheric origin. Following this, two of the animals were sacrificed and samples of different fat, muscle, and organ tissues were collected. In Phase 2 the remaining two animals were fed grass silage from a field which had a history of repeated sewage sludge applications. During the last 10 days of the 27-day feeding period, the dietary uptake and fecal excretion of PCDD/Fs were again quantified, after which these two animals were also sacrificed and sampled. The dietary absorption of the PCDD/Fs in the nonlactating cows agreed well with values reported in Part I of this series for lactating cows. In the two animals sacrificed at the end of Phase 1 that were close to a contaminant steady state, the lipid-normalized concentrations were similar in almost all tissues. The exceptions were the liver, and to a lesser extent the lungs and the spleen, which had higher levels; and the degree of elevation increased with the degree of chlorination of the PCDD/Fs. During Phase 2, the animals' body burden of several of the PCDD/F congeners increased markedly. The tissue analyses indicated that the chemicals were initially sequestered primarily in the liver, from where they were redistributed to the other tissues and organs. The rate of redistribution was related to the perfusion of the organ/tissue and decreased in the order lung>spleen>kidney>muscle>fat tissue. The rate of redistribution also decreased with increasing degree of chlorination of the PCDD/F congeners. Whereas virtually all of the 1,2,3,7,8-Cl(5)DD taken up during Phase 2 had been deposited in fat tissue by the end of the 27-day feeding period, three-quarters of the Cl(8)DD was still in the liver.     

Determination of melengestrol acetate in feedstuffs with liquid chromatographic preparatory column cleanup and quantitative analysis

Melengestrol acetate (MGA) is determined by liquid chromatography using a fraction from preparatory LC as a means of sample cleanup for feedstuffs, both dry and liquid. Dry ground feed is Soxhlet extracted with hexane and passed through a 2% deactivated alumina column for initial cleanup. The eluate is evaporated, redissolved in methanol, filtered, and injected onto a preparatory LC column. The fraction containing MGA is separated from the remaining matrix, evaporated to dryness, dissolved in methanol, and quantitated by LC analysis. Liquid supplements are extracted in methanol, and the extract is evaporated to near dryness. The residue is diluted with water, extracted with chloroform, passed through sodium sulfate, and evaporated to dryness. The remaining sample is dissolved in methanol prior to preparative LC and quantitative LC. Recoveries for 2 laboratory-fortified commercial feeds, one dry and one liquid, containing 0.39 and 0.40 mg/lb, were 98.3% +/- 4.4 and 95.8% +/- 4.3, respectively. Results compare favorably with existing methods. Up to a 4-fold time savings was realized by this method without automation.     

Identification of species in animal feedstuffs by polymerase chain reaction--restriction fragment length polymorphism analysis of mitochondrial DNA

Restriction site analysis of Polymerase Chain Reaction (PCR) products of cytochrome b mitochondrial DNA was applied to identify species in meat meal and animal feedstuffs. PCR was used to amplify a variable region of cytochrome b mitochondrial DNA gene. Species differentiation was determined by digestion of the obtained 359 bp amplicon with restriction enzymes, which generated species-specific electrophoresis patterns; the sequencing of PCR products was used as confirming analysis. PCR-RFLP analysis revealed the presence of meat meal in animal feedstuffs and distinguished species of interest. The results supported the application of the method in control measures which should be adopted for meat-meal-based animal feed, as suggested by EU law. As a technical improvement, to simplify the analysis, the number of enzymes presented in this study for the detection of different species was smaller than others described in the literature; discrimination between ruminant and nonruminant species and between mammalian and poultry species was possible with few digestions.     

Alpha-tocopherol content in 62 edible tropical plants.

Vitamin E was determined by the high-performance liquid chromatography (HPLC) method. All the plants tested showed differences in their alpha-tocopherol content and the differences were significant (p < 0.05). The highest alpha-tocopherol content was in Sauropus androgynus leaves (426.8 mg/kg edible portion), followed by Citrus hystrix leaves (398.3 mg/kg), Calamus scipronum (193.8 mg/kg), starfruit leaves Averrhoa belimbi (168.3 mg/kg), red pepper Capsicum annum (155.4 mg/kg), local celery Apium graveolens (136.4 mg/kg), sweet potato shoots Ipomoea batatas (130.1 mg/kg), Pandanus odorus (131.5 mg/kg), Oenanthe javanica (146.8 mg/kg), black tea Camelia chinensis (183.3 mg/kg),papaya Carica papaya shoots (111.3 mg/kg), wolfberry leaves Lycium chinense (94.4 mg/kg), bird chili Capsicum frutescens leaves (95.4 mg/kg), drumstick Moringa oleifera leaves (90.0 mg/kg), green chili Capsicum annum (87 mg/kg), Allium fistulosum leaves (74.6 mg/kg), and bell pepper Capsicum annum (71.0 mg/kg). alpha-Tocopherol was not detected in Brassica oleracea, Phaeomeria speciosa, Pachyrrhizus speciosa, Pleurotus sajor-caju, and Solanum melongena.