Comparison of two direct methods for the determination of fatty acids in infant feces

We have validated and compared two direct methods for the determination of fatty acids in feces by capillary gas chromatography. Method I consisted of esterification of fatty acids using acetyl chloride. Method II used boron trifluoride-methanol as esterification reagent. The two methods were assayed with and without previous freeze-drying of the fecal sample. We found that the two methods could be carried out without sample freeze-drying. Precision and recovery rates were determined and the results were satisfactory. Both methods gave similar results, but Method II has certain advantages over Method I, such as speed, safety, and better recovery rates.     

Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP)

Background  

The microbiota of an animal's intestinal tract plays important roles in the animal's overall health, productivity and well-being. There is still a scarcity of information on the microbial diversity in the gut of livestock species such as cattle. The primary reason for this lack of data relates to the expense of methods needed to generate such data. Here we have utilized a bacterial tag-encoded FLX 16s rDNA amplicon pyrosequencing (bTEFAP) approach that is able to perform diversity analyses of gastrointestinal populations. bTEFAP is relatively inexpensive in terms of both time and labor due to the implementation of a novel tag priming method and an efficient bioinformatics pipeline. We have evaluated the microbiome from the feces of 20 commercial, lactating dairy cows.     

In vivo total antioxidant capacity: comparison of different analytical methods

Several methods have been developed to measure the total antioxidant capacity of a biological sample. The use of peroxyl or hydroxyl radicals as pro-oxidants in the oxygen radical absorbance capacity (ORAC) assay makes it different and unique from the assays that involve oxidants that are not necessarily pro-oxidants. An improvement in quantitation is achieved in the ORAC assay by taking the reaction between substrate and free radicals to completion and using an area-under-curve technique for quantitation compared to the assays that measure a lag phase. The interpretation of the changes in plasma or serum antioxidant capacity becomes complicated by the different methods used in detecting these changes. The interpretation also depends upon the conditions under which the antioxidant capacity is determined because the measurement reflects outcomes in a dynamic system. An increased antioxidant capacity in plasma or serum may not necessarily be a desirable condition if it reflects a response to increased oxidative stress. Similarly, a decrease in plasma or serum antioxidant capacity may not necessarily be an undesirable condition if the measurement reflects decreased production of reactive species. Because of these complications, no single measurement of antioxidant status is going to be sufficient, but a "battery" of measurements, many of which will be described in Forum articles, will be necessary to adequately assess oxidative stress in biological systems.     

Evaluation of methods for total selenium determination in yeast

The selenium determination in biological materials by the classical fluorometric method (FM) is time-consuming and also hazardous, as it requires the destruction of the organic matrix samples with hot HNO₃/HClO₄ mixtures prior to analysis. Accordingly, commercial analytical laboratories are increasingly using faster instrumental methods; for sample digestion, avoid using HClO₄. Because of these procedural changes, the results obtained by commercial laboratories may be unreliable, especially for samples containing Se in organic forms. One such “difficult” substrate is Se yeast, which contains most of its Se as selenomethionine. To establish which methods for Se analysis and sample digestion are applicable, samples of Se yeast and of selenomethionine standards were sent to laboratories employing either flame atomic absorption spectrometry (FAAS), inductively coupled plasma-mass spectrometry (ICP-MS), or hydride generation atomic absorption spectrometry (HGAAS). The result were compared with those obtained by FM and non-destructive instrumental neutron activation analysis (INAA). ICP-MS, after microwave digestion of sample with HNO₃/H₂O₂, produced results within 5% of the expected values, as did those obtained by FM and INAA. With FAAS, acceptable results were obtained after digestion with HNO₃/HCl. With HGAAS, sample digestion with HNO₃/H₂O₂ produced values that were systematically elevated by about 10% and exhibited standard deviations of ≥10%. Thus, current methods of sample digestion are applicable for Se yeast analysis by ICP-MS and FAAS, but not by HGAAS.     

Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters

Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and then analyzed 14 to 17 days later. The V. parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (r = 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r = 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total V. parahaemolyticus, and they were more rapid and less labor-intensive.     

Prevalence of Escherichia coli O157 in cattle feeds in Midwestern feedlots

Comparisons of enrichment methods (with or without antibiotics and with or without a preenrichment step) using gram-negative (GN) broth or tryptic soy broth (TSB) were conducted with feeds inoculated with Escherichia coli O157:H7. TSB was more sensitive than GN broth, and TSB with a preenrichment step followed by TSB with antibiotics was more sensitive than plain TSB enrichment, in detecting E. coli O157 in inoculated feeds. Feed samples were collected from feed bunks from 54 feedlots to determine the prevalence of E. coli O157 in cattle feeds. TSB preenrichment followed by TSB with antibiotics and the standard GN broth enrichment were used for each feed sample. All samples underwent immunomagnetic separation and were plated onto sorbitol MacConkey agar with cefixime and potassium tellurite. Identification of E. coli O157 was based on indole production, positive latex agglutination for O157 antigen, API 20E test strip results, PCR for the eaeA gene, and the presence of at least one Shiga toxin. E. coli O157 was detected in 52 of 504 feed samples (10.3%) by using GN broth enrichment and in 46 of 504 feed samples (9.1%) by using TSB followed by TSB supplemented with cefixime and vancomycin. E. coli O157 was detected in 75 of 504 feed bunk samples (14.9%) by one or both methods. There was no correlation between E. coli O157 prevalence and generic coliform counts in feeds. The prevalence of E. coli O157 in cattle feed warrants further studies to increase our knowledge of the on-farm ecology of E. coli O157 in order to develop strategies to prevent food-borne disease in humans.     

Concentration and prevalence of Escherichia coli O157 in cattle feces at slaughter

The concentration and prevalence of Escherichia coli O157 in cattle feces at the time of slaughter was studied over a 9-week period from May to July 2002. Fecal samples (n = 589) were collected from the rectums of slaughtered cattle, and the animal-level prevalence rate was estimated to be 7.5% (95% confidence interval [CI], 5.4 to 9.6%) while the group prevalence was 40.4% (95% CI, 27.7 to 53.2%). Of the 44 infected animals detected, 9% were high shedders that contained E. coli O157 at concentrations of >10(4) CFU g(-1). These 9% represented >96% of the total E. coli O157 produced by all animals tested. All isolates possessed the vt(2) gene, 39 had the eaeA gene, and a further five had the vt(1) gene also. The presence of high-shedding animals at the abattoir increases the potential risk of meat contamination during the slaughtering process and stresses the need for correctly implemented hazard analysis and critical control point procedures.     

Comparison of two methods for recovery of ovarian oocytes from slaughter cattle

When aspiration was used, 80 oocytes were recovered from 185 ovarian follicles, whereas 155 oocytes were recovered by rupturing 154 isolated follicles (P < 0.01).Two oocytes were found in each of three isolated follicles, three oocytes in one follicle and no oocytes in four follicles.Of the oocytes recovered by aspiration, 45% were morphologically normal, whereas 63.2% morphologically normal oocytes were recovered from isolated follicles (P < 0.01).     

Comparison of two methods for one-step in-straw thawing and direct transfer of cattle blastocysts

The aim of this experiment was to evaluate the feasibility of dilution of the cryoprotectant by a sucrose solution in the straw followed by direct transfer without any selection of the embryos. A comparison was made between the method described by Renard et al. (7) and a method slightly modified from that published by Leibo (8). The feasibility is proved by a mean pregnancy rate of 41.4%. Although the difference was not statistically significant, the method described by Renard et al. (7) led to a higher pregnancy rate (44.7%) than that of Leibo (8) (38.5%).     

Evaluation of variant identification methods for whole genome sequencing data in dairy cattle

Background

Advances in human genomics have allowed unprecedented productivity in terms of algorithms, software, and literature available for translating raw next-generation sequence data into high-quality information. The challenges of variant identification in organisms with lower quality reference genomes are less well documented. We explored the consequences of commonly recommended preparatory steps and the effects of single and multi sample variant identification methods using four publicly available software applications (Platypus, HaplotypeCaller, Samtools and UnifiedGenotyper) on whole genome sequence data of 65 key ancestors of Swiss dairy cattle populations. Accuracy of calling next-generation sequence variants was assessed by comparison to the same loci from medium and high-density single nucleotide variant (SNV) arrays.

Results

The total number of SNVs identified varied by software and method, with single (multi) sample results ranging from 17.7 to 22.0 (16.9 to 22.0) million variants. Computing time varied considerably between software. Preparatory realignment of insertions and deletions and subsequent base quality score recalibration had only minor effects on the number and quality of SNVs identified by different software, but increased computing time considerably. Average concordance for single (multi) sample results with high-density chip data was 58.3% (87.0%) and average genotype concordance in correctly identified SNVs was 99.2% (99.2%) across software. The average quality of SNVs identified, measured as the ratio of transitions to transversions, was higher using single sample methods than multi sample methods. A consensus approach using results of different software generally provided the highest variant quality in terms of transition/transversion ratio.

Conclusions

Our findings serve as a reference for variant identification pipeline development in non-human organisms and help assess the implication of preparatory steps in next-generation sequencing pipelines for organisms with incomplete reference genomes (pipeline code is included). Benchmarking this information should prove particularly useful in processing next-generation sequencing data for use in genome-wide association studies and genomic selection.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-948) contains supplementary material, which is available to authorized users.     

Evaluation of two simplified Life Cycle assessment methods

Goal, Scope and Background  Two methods of simplified LCA were evaluated and compared to the results of a quantitative LCA. These are the Environmentally responsible product assessment matrix developed by Graedel and Allenby and the MECO-method developed in Denmark. Methods  We used these in a case study and compared the results with the results from a quantitative LCA. The evaluation also included other criteria, such as the field of application and the level of arbitrariness. Results and Discussion  The MECO-method has some positive qualities compared to the Environmentally responsible product assessment matrix. Examples of this are that it generates information complementary to the quantitative LCA and provides the possibility to consider quantitative information when such is available. Some of the drawbacks with the Environmentally responsible product assessment matrix are that it does not include the whole lifecycle and that it allows some arbitrariness. Conclusions  Our study shows that a simplified and semi-quantitative LCA (such as the MECO-method) can provide information that is complementary to a quantitative LCA. In this case the method generates more information on toxic substances and other impacts, than the quantitative LCA. We suggest that a simplified LCA can be used both as a pre-study to a quantitative LCA and as a parallel assessment, which is used together with the quantitative LCA in the interpretation. Recommendations and Outlook  A general problem with qualitative analyses is how to compare different aspects. Life cycle assessments are comparative. The lack of a quantitative dimension hinders the comparison and can thereby hinder the usefulness of the qualitative method. There are different approaches suggested to semiquantify simplified methods in order to make quantitative comparisons possible. We think that the use of fabricated scoring systems should be avoided. If quantitative information is needed, one should consider performing a simplified quantitative LCA instead.     

The effectiveness of gender determination using polymerase chain reaction and radioimmunoassay methods in cattle

The aim of this study was to use polymerase chain reaction (PCR) by amplifying DNA from bovine (Bos taurus) fetal cells recovered through uterine puncture and subsequent amniotic fluid aspiration and to compare the effectiveness of the PCR method with amniotic dihydrotestosterone (DHT) levels in gender determination. Amniotic DHT levels between sexes were significantly higher in males than in females in all periods except the period 91 to 120 d. The differences among the amniotic DHT levels at different gestation periods (61 to 90, 91 to 120, 121 to 150, 151 to 180, 181 to 210 d) were not significant in females but were significant in males in the period 61 to 90 d compared with three other periods. Sensitivity was equal to 97.8% (95% CI = 88.2% to 99.6%), and specificity was equal to 85.4% (95% CI = 80.0% to 97.6%). These two values correspond with a cutoff of DHT in amniotic fluid. Distributions of the two sex groups were classified according to the 192.1 pg/mL cutoff value. A total of 93 amniotic fluid samples were examined by PCR analysis. The sex determination of 91 samples by PCR and electrophoresis was in agreement with the visual sexes of the fetuses. In two amniotic fluid samples, DNA was not isolated, and thus no sex determination was made. Fetal gender was correctly identified by PCR in 44 of 45 males and in 47 of 48 females. In PCR, one band (at the length of 102 bp) and two bands (at the lengths of 102 and 226 bp) were observed respectively for female and male fetuses. It may be concluded that the levels of amniotic DHT and PCR might be used for embryo sexing in pregnant cows.     

Comparison of gas accumulation profiles of several feeds using manual or automated gas production methods

The relationship of metabolizable energy (ME) content of the diet to gas production measured by the Hohenheim gas test (HGT) has been studied intensively. However, the HGT is being replaced by automatic systems like the automated pressure evaluation system (APES) and comparison with the HGT method is required before ME estimation can be automated. This study compared the two different gas production methods (HGT and the APES) with regard to the cumulative gas profile. With the APES method, the release of gas may occur at any time, assuming fixed amounts of gas being released for each venting, after reaching fixed values of pressure. With the HGT method manual readings are performed at defined time points. For comparison purposes, gas production was calculated on the basis of ml/200 mg dry matter (DM), as usual in the HGT method. For 11 feeds analyzed (grass silage, meadow hay, fresh red clover, fresh birdsfoot trefoil, whole-crop oat silage, maize stover and ear maize, dairy compound feed and soybean meal) the APES method produced on average 5.5 ml (range 1.1–8.3 ml/200 mg DM) less gas on average compared to the HGT method (0–120 h incubation time). Reasons for the differences may be related to the measurement conditions of each method itself. The ratio of sample size to rumen fluid (mg/ml) is 20:1 in the HGT method and 100:1 in the APES method, which may have influenced the colonization rate and contributed to a larger lag phase in APES. The estimates are based on two runs, which were performed on different days, with the APES method generating a large run effect. In conclusion, the amount of gas produced using the APES method deviated consistently from the amounts of gas produced by the HGT method in a large range of gas production (30–72 ml/200 mg DM). Using the laboratory protocol as proposed by each method, the suggested mathematical correction of the gas measured through APES appears applicable for gas production after 24 h, needing a larger number of samples to prove its efficacy.     

Evaluation of angiosperm and fern contributions to soil organic matter using two methods of pyrolysis-gas chromatography-mass spectrometry

Background

Ferns are an important plant group, and older phylogenies of non-polypod ferns contain relatively high concentrations of aliphatic leaf waxes, lignins, and tannins that could contribute to soil organic matter (SOM) biochemistry and stability.

Methods

Pyrolysis gas-chromatography mass-spectrometry (py-GC/MS) analyzes biochemical fragments which can be related to lignin, polysaccharide, lipid, nitrogen (N)-bearing, non-lignin aromatics, and phenol source compounds. Thermochemolysis using tetramethylammonium hydroxide (TMAH) combined with py-GC/MS improves detection of lignin, cutin, and suberin-derived compounds. To examine the advantages and disadvantages of both methods for characterizing plant and soil biochemistry, we characterized non-polypod and polypod fern and angiosperm live tissues, roots and soils from the Kohala Mountains, Hawaii.

Results

Py-GC/MS provided a broad biochemical overview of compound groups including lignin, polysaccharide, lipid, N-bearing, non-lignin aromatics and phenol groups while TMAH-py-GC/MS detailed lignin units and fatty acids at the expense of the other categories. TMAH-py-GC/MS provided more detailed data on lignin, cutin, suberin and tannin-derived compounds. Both methods detected differences in lignin units between species, although p-coumaric and ferulic acids, predominantly found in ferns, were only observed with TMAH-py-GC/MS.

Conclusions

Both py-GC/MS and TMAH-py-GC/MS are methods to detect compound-specific plant biomarkers, but are most useful when combined for their complimentary results.     

Evaluation of two methods to estimate and monitor bird populations

Background

Effective management depends upon accurately estimating trends in abundance of bird populations over time, and in some cases estimating abundance. Two population estimation methods, double observer (DO) and double sampling (DS), have been advocated for avian population studies and the relative merits and short-comings of these methods remain an area of debate.

Methodology/Principal Findings

We used simulations to evaluate the performances of these two population estimation methods under a range of realistic scenarios. For three hypothetical populations with different levels of clustering, we generated DO and DS population size estimates for a range of detection probabilities and survey proportions. Population estimates for both methods were centered on the true population size for all levels of population clustering and survey proportions when detection probabilities were greater than 20%. The DO method underestimated the population at detection probabilities less than 30% whereas the DS method remained essentially unbiased. The coverage probability of 95% confidence intervals for population estimates was slightly less than the nominal level for the DS method but was substantially below the nominal level for the DO method at high detection probabilities. Differences in observer detection probabilities did not affect the accuracy and precision of population estimates of the DO method. Population estimates for the DS method remained unbiased as the proportion of units intensively surveyed changed, but the variance of the estimates decreased with increasing proportion intensively surveyed.

Conclusions/Significance

The DO and DS methods can be applied in many different settings and our evaluations provide important information on the performance of these two methods that can assist researchers in selecting the method most appropriate for their particular needs.     

Evaluation of bull fertility in dairy and beef cattle using cow field data

A successful outcome to a given service is a combination of both male and female fertility. Despite this, most national evaluations for fertility are generally confined to female fertility with evaluations for male fertility commonly undertaken by individual breeding organisations and generally not made public. The objective of this study was to define a pertinent male fertility trait for seasonal calving production systems, and to develop a multiple regression mixed model that may be used to evaluate male fertility at a national level. The data included in the study after editing consisted of 361,412 artificial inseminations from 206,683 cow-lactations (134,911 cows) in 2,843 commercial dairy and beef herds. Fixed effects associated with whether a successful pregnancy ensued (pregnant = 1) or not (pregnant = 0) from a given service were year by month of service, day of the week, days since calving, cow parity, level of calving difficulty experienced, whether or not the previous calving was associated with perinatal mortality, and age of the service bull at the date of insemination. Non-additive genetic effects such as heterosis and recombination loss as well as inbreeding level of the service bull, dam or mating were not associated with a successful pregnancy; there was no difference in pregnancy rate between fresh or frozen semen. Random effects included in the model were the additive genetic effect of the cow, as well as a within lactation and across lactation permanent environmental effect of the cow; pedigree group effects based on cow breed were also included via the relationship matrix. Temporal differences in the AI technician and service bull were also included as random effects. A difference in five percentage units in male fertility was evident between the average effects of different dairy and beef breeds. The correlation between raw pregnancy rates for bulls with more than 100 services (n = 431) and service bull solutions from the mixed model analysis was 0.66. The correlation between the raw pregnancy rates of 288 technicians with more than 100 services and their respective solutions from the mixed model was 0.35. These low to moderate correlations suggest considerable re-ranking among both service bulls and technicians and suggest possibly a benefit of using a statistical model to better estimate the performance of both service bulls and technicians.