Species-specific PCR for the identification of ruminant species in feedstuffs

A polymerase chain reaction (PCR) method based on the nucleotide sequence variation in the 12S ribosomal RNA mitochondrial gene has been developed for the specific identification of bovine, ovine and caprine DNAs in feedstuffs. The primers designed generated specific fragments of 84, 121 and 122pb length for bovine, ovine and caprine species, respectively. The specificity of the primers designed was tested against 30 animal species including mammals, birds and fish, as well as eight plant species. Analysis of experimental feedstuffs demonstrated that 0.1% of raw and heated bovine, ovine or caprine tissues can be easily detected using the species-specific primers developed. The performance of this method is not affected by prolonged heat treatment, and consequently it could be very useful to verify the origin of the raw materials in products submitted to denaturing technologies, for which other methods cannot be applied.     

Feed protein fractions in various solvents of ruminant feedstuffs

The classical protein fractions, ie albumins, globulins, prolamins, glutelins and insoluble (or structural) proteins were evaluated in selected feedstuffs. Solvents used for classical protein fractionation were: dilute salt (1 M NaCl) in phosphate buffer (pH 7·0), aqueous alcohol (70% ethanol) and dilute alkali (0·05 M NaOH), with the subsequent separation of albumins and globulins by dialysis. In addition, crude protein content, soluble non-protein nitrogen, total true protein and soluble true protein in McDougall's buffer from the same feedstuffs were determined. The soluble non-protein N contributed only a small portion of the total N in feedstuffs. Total true protein was approximately 8–10% for energy feeds, and then increased to 30% for plant protein sources, while the soluble true protein was about the same for energy feeds and plant protein sources. The major proportion of protein in plant protein sources was in the form of globulins, followed by albumins and lesser amounts of glutelins and prolamins. In contrast, the major proportion of protein in cereals was in the form of prolamins and glutelins, followed by globulins and albumins. © 1997 SCI.     

Total and ileal digestible tryptophan contents of feedstuffs for broiler chickens

Reliable values of total and digestible tryptophan in components of feed formulation matrices are needed because tryptophan is often the third limiting amino acid in practical poultry diets. However, tryptophan is oxidatively destroyed during acid hydrolysis in routine amino acid analysis and its determination requires a separate analytical procedure. The variability in contents and apparent ileal digestibility for 6‐week‐old broiler chickens of tryptophan in 74 samples representing 24 feedstuffs are presented in this paper. The average ileal tryptophan digestibility coefficient in wheat was 0.83, in sorghum and triticale 0.75, maize 0.71, soybean meal 0.84, sunflower meal 0.81, canola meal 0.78 and cottonseed meal 0.75. Among the grain legumes, tryptophan in lupins was better digested than that in chickpeas, fababeans and field peas. Among the animal protein meals, the tryptophan digestibility coefficients in fish meal (0.77) and blood meal (0.84) were substantially higher than those in meat meal (0.62), meat‐and‐bone meal (0.63) and feather meal (0.52). Marked variations in tryptophan digestibility were also observed among samples of fish meal, meat‐and‐bone meal and meat meal, highlighting significant batch‐to‐batch differences. For most feedstuffs, considerable variability was observed in the tryptophan concentrations, but such variations were not reflected in digestibility coefficients. Copyright © 2006 Society of Chemical Industry     

Nitrogen solubility in three solvents and in situ protein degradability of ruminant feedstuffs

Feedstuffs commonly fed to ruminants were assayed for nitrogen solubility by using McDougall's buffer, 0·02 M sodium hydroxide (NaOH), or 0·15 M sodium chloride (NaCl) as solvents. In addition, in situ dry matter and protein disappearance from the same feedstuffs were determined using the nylon bag technique in ruminally cannulated sheep for varying times of incubation. The mean effective nitrogen disappearance ranged from 235 for maize to 894 g kg⁻¹ of total N for lupins, and dry matter disappearance from 240 for meat and bone meal to 793 g kg⁻¹ for lupins. Protein solubility was lowest (<10% of total N) for oilseed by-products and animal and fish by-products, intermediate (15–30% of total N) for some cereals and highest (35–45% of total N) for wheat varieties and plant protein sources. Furthermore, solubility and degradability data for various feed proteins are presented which demonstrate the variability in solubility and degradability of ruminant feedstuffs due to protein type or processing. © 1997 SCI.     

Comparison of sheep and red deer rumen fluids for assessing nutritive value of ruminant feedstuffs

Four forages (alfalfa hay, barley straw, maize silage and beet pulp), four cereal grains (barley, maize, sorghum and wheat) and four shrubs (Calluna vulgaris, Erica australis, Cytisus cantabricus and Genista occidentalis) were incubated using rumen fluids from sheep and red deer, to examine differences in in vitro fermentation rates and ruminal parameters. For the forages and cereal grains, results suggest few differences between species in parameters related to gas production, rates of fermentation, organic matter disappearance and extent of degradation. Deer showed slightly better results for shrubs than sheep did. Disappearance of neutral detergent fibre was generally greater when the rumen fluid was derived from red deer (P < 0.05). On the other hand, ammonia‐N concentration and total volatile fatty acids (VFAs) were higher in sheep (P < 0.05). Molar proportions of the major VFAs showed significant differences (P < 0.05) associated with the species of the inoculum donor and suggest that fermentation pathways might have been more efficient in red deer. The estimated amount of methane was higher in sheep, regardless of the substrate incubated (P < 0.001). The results indicate that although the sheep could be valid as a model to assess the nutritive value of good quality feedstuffs for red deer, it would fail to offer reliable information on non‐conventional, low‐quality feeds such as shrubs. Copyright © 2005 Society of Chemical Industry     

Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for sensitive detection of prohibited ruminant proteins in feedstuffs

Regulations aimed to control the epidemic of bovine spongiform encephalopathy have banned the use of certain animal products, i.e., ruminant meat and bone meals, in ruminant animal feeds. A sensitive enzyme-linked immunosorbent assay has been developed to detect prohibited bovine and ovine muscles in feedstuffs. The assay utilizes a pair of monoclonal antibodies (MAbs) against skeletal troponin I (TnI). MAb 5G9, specific to bovine and ovine TnI, was used as the capture antibody and the biotin-conjugated MAb 2G3, reacting to all heterologous TnI, was used as the detection antibody. Quantitative procedures were applied to samples containing 5, 0.5, and 0.05% (wt/wt) of heat-treated (132 degrees C/2 bar, 2 h) bovine and ovine meat meals in three different feeds, coexisting with porcine, chicken, or turkey meat meal. The presence of these nonprohibited species did not affect the detection of bovine and ovine meat meals in the feed samples (P > 0.05). Quantitative determinations of extractable bovine and ovine TnI, with a detection limit of 5.0 and 4.0 ng/ml, respectively, were achieved when the matching feed matrixes were used in the calibration curves. This new assay provides a rapid and reliable way to detect animal protein products containing a trace amount of bovine or ovine muscle tissue in feedstuffs.     

In vitro methods for measuring the dry matter digestibility of ruminant feedstuffs: comparison of methods and inoculum source

We conducted this study to evaluate the new in vitro system, DAISY", to determine dry matter (DM) digestibility in ruminant feedstuffs. Results from the DAISY" were compared to those obtained by the traditional Tilly and Terry method. The traditional method buffer was used for both methods. We also compared two sources of rumen inoculum from sheep and dairy cows. Seventeen different feeds were tested, grouped into roughage, concentrate, and CP supplements. The experiment was replicated on two different occasions for all feeds and the two sources of inoculum. The source of inoculum and the time at which it was collected had no effect on the in vitro DM digestibility of the feedstuffs in either of the methods. The DAISY" DM digestibility value compared well with the traditional method values for the roughage group; however, for some feedstuffs in the concentrate and CP supplement groups, the DAISY" values were significantly higher than the traditional method values. Regression analysis of the feeds that resulted in similar values with the two methods revealed that the DAISY" method can be used to predict in vitro digestibility with relatively small variation.     

A study of the variability of the endogenous energy output by adult roosters and a determination of the available energy of nine different feedstuffs

The variability of energy excretion by adult roosters starved for 24 h or force-fedvarying levels of dextrose following a 24-h fast was studied in three separate balance trials. A fourth balance trial was conducted to determine the available energy content of several feedstuffs. Dry matter and energy excretion were measured in the first three trials and available energy was determined as true metabolisable energy (TME) for adult roosters and as apparent metabolisable energy (AME) corrected to zero nitrogen retention for growing rats and pigs in the fourth trial. A significant (^p < 0.05) positive correlation (r=0.81) between body weight (range 1.9–2.8 kg) of roosters and energy excretion was observed. Energy excretion by roosters tended to vary with the level of dextrose force-fed and the calculation of TME of dextrose produced values equal to its gross energy at high (50–60 g) levels of intake. The TME content of nine feedstuffs varying in chemical composition are given and compared with AME values determined with rats and pigs.     

Estimation of mean and median particle size of ruminant digesta

An alternative exponential equation with a parameter as an exponent was compared to a recently published exponential procedure for estimating mean and median particle size. Both equations were fit to literature values for particle size distributions of esophageal extrusa, rumen digesta, and feces. In addition, 112 observations of particle-size distributions of esophageal extrusa were fit to both equations for comparison. Both equations gave adequate fits of rumen digesta and feces, but the earlier model was biased by regular deviations from observed data for esophageal extrusa. Use of the previously published model overestimated mean particle size in esophageal extrusa from 4.5 to 10.3%. Median particle size was underestimated from 4.9 to 5.6%. Analytic solutions exist for mean and median particle sizes for the earlier model, but it is necessary to estimate numerically the mean and median particle sizes when using the proposed equation. Even though the solution for the proposed equation must be determined numerically, it will prevent bias between experimental treatments, give more accurate estimates of mean and median particle size, and result in a lower residual sums of squares. In addition, a single equation can be used to model particle size reduction throughout the digestive tract.     

Metabolisable energy values for growing chicks of some feedstuffs from Sri Lanka

Seven chick bioassays were conducted with 600 one-week-old White Leghorn cockerels to determine the zero nitrogen-corrected metabolisable energy (ME_n) values of twenty-three Sri Lankan-produced feedstuffs. The ME_n values (MJ kg^?1 dry matter) of the feedstuffs were: maize 14.28, sorghum 14.42, broken rice 12.35, rough rice 9.21, rice bran grade I 14.28, rice bran grade II 9.30, wheat feed flour 9.31, wheat bran 7.66, undetoxified cassava root meal 13.38, detoxified cassava root meal 15.92, coconut oil meal 7.46, sesame oil meal 11.48, rubber seed meal 11.25, kapok seed meal 8.75, urd bean 12.68, cassava leaf meal 7.82, local fish meal 11.97, skim milk powder 10.41, meat and bone meal 8.02, sugar filter-press mud 8.85, cocoa husk meal 7.46, Mango seed kernel meal 10.84, and tea refuse 6.02. The difference between the ME_n values of undetoxified and detoxified cassava root meal indicate that hydrocyanic acid interferes with energy utilisation in poultry.     

Towards a quantitative application of real-time PCR technique for fish DNA detection in feedstuffs

A real-time PCR method to detect fish DNA in feedstuffs was developed and optimised. A combination of primers and a Taqman-MGB probe was used to selectively amplify the fish mitochondrial 12S ribosomal RNA gene. Qualitative and also quantitative assessments were performed with different protocols: a relative quantification by a standard curve, and a ΔC_T method, by total plant DNA as endogenous controls. Method specificity was evaluated analysing 40 different tissues (mammalians, avian, fish) and flour samples. Sensitivity was evaluated by LOD (limit of detection) estimation. The designed probe–primers set showed an increased sensitivity compared to previously published PCR end point method, reaching a limit of detection of 0.2 pg of fish DNA, and showing to be a robust assay for fish DNA detection. The quantification results, based on ΔC_T method and the relative standard curve, are well reproducible in our experimental condition but, in lacking of separate pure raw materials of a tested feed, they cannot be applied for reliable and precise quantification on field samples but for now as a semi-quantitative PCR method only.     

慢消化淀粉定量测定方法的比较研究

慢消化淀粉是指在人体小肠中能完全被消化吸收,但吸收速度较慢的一类淀粉。慢消化淀粉血糖指数低,具有特殊的生理功能,是近年来的研究热点;但是慢消化淀粉的定量测定仍然没有统一的标准。用了3种常用的慢消化淀粉定量测定方法:Englyst法、Shin法、Miao法,对大米淀粉、小麦淀粉和糯米淀粉3种原料中的慢消化淀粉进行定量测定,并进行比较分析。结果表明:Miao法模拟了人体胃肠道的生理条件,具有重复性好,操作简单等明显优势,适合于谷物类原料中慢消化淀粉含量的测定;用该法测得的大米淀粉、小麦淀粉、糯米淀粉的慢消化淀粉含量分别为:41·6%、49·4%、54·4%。      

The value of different fat supplements as sources of digestible energy for lactating dairy cows

The effects of fat supplements that differed in fatty acid composition (chain length and degree of saturation) and chemical form (free fatty acids, Ca salts of fatty acids, and triacylglyceride) on digestible energy (DE) concentration of the diet and DE intake by lactating cows were measured. Holstein cows were fed a control diet [2.9% of dry matter (DM) as long-chain fatty acids] or 1 of 3 diets with 3% added fatty acids (that mainly replaced starch). The 3 fat supplements were (1) mostly saturated (C18:0) free fatty acids (SFA), (2) Ca-salts of fatty acids (CaFA), and (3) triacylglyceride high in C16:0 fatty acids (TAG). Cows fed CaFA (22.8 kg/d) consumed less DM than cows fed the control (23.6 kg/d) and TAG (23.8 kg/d) diets but similar to cows fed SFA (23.2 kg/d). Cows fed fat produced more fat-corrected milk than cows fed the control diet (38.2 vs. 41.1 kg/d), mostly because of increased milk fat percentage. No differences in yields of milk or milk components were observed among the fat-supplemented diets. Digestibility of DM, energy, carbohydrate fractions, and protein did not differ between diets. Digestibility of long-chain fatty acids was greatest for the CaFA diet (76.3%), intermediate for the control and SFA diets (70.3%), and least for the TAG diet (63.3%). Fat-supplemented diets had more DE (2.93 Mcal/kg) than the control diet (2.83 Mcal/kg), and DE intake by cows fed supplemented diets was 1.6 Mcal/d greater than by cows fed the control, but no differences were observed among the supplements. Because the inclusion rate of supplemental fats is typically low, large differences in fatty acid digestibility may not translate into altered DE intake because of small differences in DM intake or digestibility of other nutrients.     

普鲁兰酶法制备缓慢消化淀粉工艺的研究

以普通马铃薯淀粉为原料,采用普鲁兰酶对淀粉进行酶解,制备缓慢消化淀粉。在单因素试验基础上,选取酶的添加量、酶解时间、淀粉乳浓度、冷藏时间进行中心组合(Box-Benhnken)实验设计,并运用Design-Expert8.05软件对数据进行分析和优化。结果表明:缓慢消化淀粉的最佳制备工艺条件为酶的添加量为160U/m L,酶解时间为8.5h,淀粉乳浓度10%,冷藏时间为3d,在此工艺条件下,获得SDS质量浓度为18.3%,为缓慢消化淀粉的开发提供理论依据。     

原子荧光光谱法测定饲料中镉

介绍氢化物发生-原子荧光光谱法对饲料中镉的定量分析方法，样品干法灰化后用硫酸溶解，以二硫脲—四氯化碳去除离子干扰，最佳工作条件下，该法测定线性范围0～70ng／mL，加标回收率为90％～103％，RSD为2．24％～3．41％，最低检测限为0．15ng／mL，标准曲线相关系数为0．9996，该法具有精密度高、准确性好、检出限低等优点。     

Forage plant cyclitols are readily fermented by the ruminant

In an in vitro rumen fermentation system the plant cyclitols, inositol, pinitol and quebrachitol were degraded within 48 h with gas evolution, an index of fermentation, comparable to that from mannitol and cellulose. Most of the cyclitol ring carbons appeared as volatile fatty acids (acetate, 1.2–1.5 mol; propionate, 0.8–1.0 mol). Their value as an energy source for the ruminant can thus be equated with soluble sugars, in marked contrast to their role in monogastric animal nutrition. They may be important in dry-season tropical pastures because of their high levels in water-stressed legumes.     

Determination of sulphadimidine in animal feedstuffs by an enzyme-linked immunoassay

An enzyme immunoassay technique for the determination of sulphadimidine in animal feedstuffs has been developed. The antibody showed limited cross-reactivity with other drugs, including sulphonamides, used as feed additives. Using spiked samples recoveries of 80-88% were obtained. The limit of detection of the assay was 70 ng/g.     

原子荧光光谱法测定饲料中的硒

介绍了氢化物发生——原子荧光光谱法对饲料中硒的定量分析方法,研究了电流、负高压、原子化温度对检出限的影响。最佳工作条件下,该法测定线性范围0～100ng/mL,加标回收率为98.9%～101.2%,RSD为1.206%～2.338%,最低检测限为0.4ng/mL,标准曲线相关系数为0.9997。