Flash glucose monitoring system (FGMS) is an improved subset of continuous glucose monitoring with a recognized effectiveness on glycemic control, though validation in patients with Liver Cirrhosis (LC) is lacking. To evaluate the accuracy of FGMS in patients with Type 2 Diabetes Mellitus (DM) and LC, a prospective, case-control study was performed in 61 ambulatory patients with LC and DM  or DM . During 14 days, patients performed 4 assessments per day of self-monitoring of blood glucose  followed by FGMS scanning. There were 2567 paired SMBG and FGMS values used in the accuracy analysis, with an overall mean absolute relative difference (MARD) of 12.68% in the LC group and 10.55% in the control group . In patients with LC, the percentage of readings within Consensus Consensus Error Grid analysis Zone A and A + B were 80.36% and 99,26%, respectively. Sensor clinical accuracy was not affected by factors such as body mass index, age, gender, Child-Pugh score or edematoascitic decompensation. This is the first study to approach FGMS clinical accuracy in LC, revealing a potential usability of this system to monitor glycemic control in this population. The interplay between DM or even impaired glucose tolerance and Chronic Liver Disease (CLD) is now well established in the literature7. Even though DM is a well-recognized metabolic risk factor for CLD9, the liver also plays a primary role in carbohydrate metabolism, thus DM can also occur as a result of CLD progression9. In fact, among patients with liver cirrhosis (LC), the prevalence of DM ranges from 7 to 60%, according to different authors7. Moreover, DM increases the risk of cirrhosis complications and mortality by liver failure6.Type 2 Diabetes Mellitus (DM) has been considered a Public Health challenge due to its significant impact on mortality and morbidity worldwide11. Despite the limitations described in the literature, the current options for their glycemic control monitoring are the same as the recommended for DM population in general12.Diagnosis and management of DM in patients with CLD can be extremely challenging13 and estimates the average glycemic status over the last 3 months7. This measurement frequently underestimates glycemic control in LC, probably as a result of shortened red blood cells half-life due to hypersplenism, blood loss into the gastrointestinal tract or iron deficiency15. Fructosamine also provides an estimation of average blood glucose during the past 2 weeks15. Since it represents stable ketoamines formed from glycated plasmatic proteins, this may be potentially altered by hypoalbuminemia and hyperbilirubinemia, commonly observed in CLD16. Glycated albumin is fructosamine main fraction, therefore it has similar limitations7. CLD-HbA1c formula suggested by Koga et al.17 combines HbA1c with glycated albumin. Although it appears to be a promising tool due to its high correlation with estimated HbA1C levels, there is insufficient evidence to support its use in clinical practice17.Glycated Hemoglobin (HbA1c) is routinely used as a standard measure for long-term glucose control7. It provides an accurate estimation of the real blood glucose control and is not affected by the above mentioned limitations19. However, there are a few physiologic and pharmacologic factors that may interfere with glucose readings20. Moreover, SMBG relies on meter accuracy, patients’ adherence and may not reflect glycemic fluctuations12.Self-monitoring of blood glucose (SMBG) has been suggested in CLD6. Flash glucose monitoring system (FGMS) is a subset of CGM, with the advantage of not requiring user calibration and displaying the information using graphs and trend arrows21. It is indicated for individuals with DM over 4 years old, including pregnant women, and has a proven impact on adherence, glycemic control and DM prognosis24.Continuous glucose monitoring (CGM) is a method based on interstitial glucose measurement through a cutaneous sensor19, which are easily undervalued by standard monitoring tools. Therefore, some authors defend the potential applicability of CGM in CLD, since it is not conditioned by the limitations described for standard glucose monitoring methods19. However, there is insufficient literature about the utility of CGM in these population, particularly the new subset FGMS, which has not been validated as a suitable option for monitoring DM in patients with LC.The blood glucose dynamics in CLD has been scarcely studied. Even so, and especially in LC, marked glycemia fluctuations appear to be frequent, with a higher risk of severe hypoglycemic events, postprandial hyperglycemia and nocturnal hypoglycemiaA prospective, case-control and single-center study was performed in ambulatory patients with clinical and/or histological diagnosis of LC and analytical diagnosis of DM, evaluated between January and October 2018 at the Gastroenterology Department of Braga Hospital.Patients meeting any of the following criteria were excluded: active alcohol abuse, oncological disease (particularly hepatocellular carcinoma), portal vein thrombosis, chronic pancreatitis, congestive heart failure, chronic kidney disease, Human Immunodeficiency Virus infection, Mycobacterium tuberculosis and Hepatitis C virus (HCV) infection under pharmacological treatment, corticosteroids therapy, extended hospitalization during the study period and psychic/cognitive state alteration.A sample size of 79 was sought, considering the criteria of 95% Confidence Level and 5% Confidence Interval applied by the Creative Research Systems software, available online. Since this sample could not be achieved, in order to minimize the loss of representativeness, a control group was created with patients with DM evaluated at the Endocrinology Department of Braga Hospital, without evidence of CLD . Patients were paired in a ratio 1:1, using the tool “propensity score matching” from Statistical Package for the Social Sciences , according to predefined baseline characteristics , selected to exclude any confounding factors. The exclusion criteria previously described for the LC group were also applied.Medical records were reviewed. The following parameters were considered for analysis: sociodemographic , clinical  score, edematoascitic decompensation, Body Mass Index (BMI), comorbidities and medication) and recently collected biochemical data .Written informed consent was obtained from all patients. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Braga Hospital .25. As recommended by Messer et al.26, in order to enhance sensor skin adhesion throughout device lifespan, kinesiology tape was applied over each participant’s sensor. It protects against water, increased perspiration and movement.Subcutaneous interstitial glucose levels were monitored using the FGMS FreeStyle Libre system . This tool includes a reader device and a small disposable sensor, applied on the back of the upper arm for up to 14 days, according to the manufacturer’s direction. It is factory-calibrated and has no automatic alarms. Data are transferred to the reader when it is brought into close proximity to the sensor, displaying current sensor glucose level. For a complete 24-hour glycemic profile, a scan once every 8 hours was required. Data are automatically stored on the sensor and can be uploaded from the reader, using the device software to generate summary glucose reports. Four sensor lots were used throughout the study, which is sufficient to demonstrate the performance of reagent systems across multiple production lotsSMBG was achieved by finger-prick testing of capillary blood using the same reader of FGMS , in order to reduce the variability between devices. Glucose test strips (FreeStyle Precision) and lancets (Abbott Single Fire) were provided to the participants.27 by FGMS sensor scanning. For each patient, measurements were scheduled upon waking, 2 hours after the beginning of lunch, 2 hours after the beginning of dinner and at bedtime. Though results were automatically stored and uploaded from the reader at the end of the study, individuals were also asked to register the data on a given table. Patients were advised to maintain their established diabetes management plan.There were two scheduled in-clinic visits. At the first visit (day 1), baseline HbA1c (HbA1cD1) was collected and 1 FGMS sensor per participant was inserted. Throughout the next 14 days, participants were asked to perform at least 4 assessments per day of SMBG, each followed 5 minutes laterA second visit (day 15) was programmed to deliver all the materials and upload the results. Between in-clinic visits, patients were contacted to clarify any doubt. Sensors that were accidentally dislodged within the first week of use were replaced. Sensors that were dislodged after that time were not replaced.Statistical analysis was performed using SPSS software , and MATLAB software (Mathworks Inc.).Descriptive data were summarized using the appropriate statistical tools, given the nature of the variables involved. Normal distribution was assessed by Kolmogorov-Smirnov test. Student t or Mann-Whitney tests were performed to compare the distribution of independent continuous variables. Spearman correlation was used to evaluate correlation between continuous variables. Chi-Square test was assessed to test the association between categorical variables. The level of significance was set at p < 0.05.25. Ancona et al.28 suggested that a value below 14% represents acceptable accuracy, a value between 14% and 18% represents intermediate accuracy and a value above 18% represents poor accuracy. Linear Regression was performed to identify any predictors of MARD.To evaluate the analytical accuracy of FGMS, Mean Absolute Relative Difference (MARD) was calculated using SMBG as a reference, according to the formula: (|SMBG − FMGS|)/SMBG × 10029.The Consensus and Clarke Error Grid Analysis (EGA) were used to assess the magnitude of clinical risk from inaccurate flash glucose readings . EGA, widely accepted tool for defining glucose meter’s accuracy, subdivides plotted results into 5 zones: A, less than 20% difference from reference values; B, difference greater than 20% but the resulting clinical decisions are uncritical; C, could cause an overcorrection of glycemia; D, represents a dangerous failure to detect and treat; E, leads to erroneous treatmentOver the study period, 62 participants were enrolled in the study, but ultimately only 61 patients were included in the analysis: 31 in LC group and 30 in the control group  were related to chronic alcohol abuse (CAA), followed by non-alcoholic steatohepatitis (NASH) in 19.4% (n = 6) and other causes  in 16.1% (n = 5). Patients were graded Child-Pugh A  or B . The median prognostic MELD-Na score was 10. Edematoascitic decompensation was evident in 32.3% (n = 10) and 58,1% (n = 18) were on diuretics (furosemide and/or spironolactone).There were 2565 paired capillary blood glucose to sensor glucose values used in the accuracy analyses. There were no unexpected adverse device effects reported. 4 participants (6.56%) experienced anticipated side effects (1 minor bleeding at sensor application and 3 mild erythema and itching at sensor insertion site). Eight sensors were accidentally dislodged prior to the full lifetime (4 sensors were replaced). No significant differences were observed between the groups regarding side effects and dislodged sensors. Eleven pairs were excluded because the reference glucose result was beyond the System’s dynamic range (40–500 mg/dL).The FGMS sensor results were highly correlated to capillary SMBG in LC patients  and in the control group . An overall MARD of 12.68% was obtained in the LC group and 10.55% in the control group, with a significant difference between the groups (p < 0.001). Sensor analytical accuracy remained acceptable across the 14 days and through different glucose levels , Child-Pugh B (p < 0.001), and edematoascitic decompensation (p < 0.001) were significantly associated with a higher MARD. Nevertheless, following linear regression analysis, only the presence of edematoascitic decompensation significantly influenced MARD value  et al.25. Nevertheless, a higher MARD was perceived in the second week and a wide range of MARD was observed in hypoglycemia. These results are not consistent in the literature. Some authors revealed a significant MARD decrease in second week of use and less accuracy during hypoglycemia31. It should be emphasized that studies are not completely comparable, either by study design or because the same percentage change in MARD in two studies may not correspond to the same glycemic variation. This also justifies MARDs wide percentage range. Therefore, the best assessment should be against our control group, which revealed the same results.Sensor analytical accuracy remained acceptable (below 14%) during the period evaluation and through different blood glucose intervals in LC, as also revealed by Bailey Although an acceptable analytical accuracy was achieved in patients with LC, MARD was persistently higher than the control group. We presumed that fluid overload could possibly conditioned glucose concentration in the interstitial fluid, since cirrhosis is characterized by a permanent hyperdynamic circulation and a fluid retention state. This study confirmed that the presence of edematoascitic decompensation has a significant impact on FGMS’s analytical accuracy, resulting in a higher MARD value and an intermediate accuracy.25. Considering that percentages in Zones A + B represent accurate or acceptable glucose results29, we confirmed FGMS clinical usability in LC. Sensor clinical accuracy was not affected by factors such as gender, age, body mass index, Child-Pugh or edematoascitic decompensation, as the percentages of readings within Consensus Error Grid Zone A + B were persistently high (99.67–100%).Sensor clinical accuracy was also attained in LC, 80.36% of results were placed in Zone A and 99.83% in Zones A + B of Consensus EGA. Data was supported by Clarke EGA and similar results were achieved in the control group. Bailey and colleagues found a very similar accuracy in the first study published approaching the usability of FGMS in 72 participants with type 1 or 2 DM: 86.70% results in Zone A and 100% in Zones A + B of Consensus EGAet al. (7%)21.Even though user acceptability and satisfaction were not formally assessed, there were no unanticipated device associated side effects and the frequency of sensor insertion site related side effects (6.56%) was similar to the one reported by Scott 34 used sample sizes of 8 to 89 participants. Equally important, more patients with advanced stages of cirrhosis should be included.The main limitation of this study was sample size, chosen mainly due to logistics and costs. Although data interpretation should have this in consideration, the design of the study with a control group minimize the loss of representativeness. Moreover, most studies approaching FGMSIn conclusion, this tool displayed a very satisfactory performance and usability in patients with LC and it sustained clinical accuracy in different stages of cirrhosis and in the presence of fluid overload, frequently observed in this population. This study provides promising preliminary evidence of sufficient performance and potential clinical usability to justify further randomized multicentric research.


To analyse serum folate levels in women of childbearing age in the Metropolitan Region (MR) of Chile.Encuesta Nacional de Salud, ENS 2016–2017), using a household-based multistage stratified random sample. Serum folate levels measured by electrochemiluminescence immunoassay in fasting venous blood samples were classified as deficient (<4·4 ng/ml), normal (4·4–20 ng/ml) or supraphysiological (>20 ng/ml).Cross-sectional design as part of the 2016–2017 National Health Survey (The MR of Chile.n 222) from the MR participated in the ENS 2016–2017.Women of reproductive age , 13·9 and 2·1–32·2 ng/ml, respectively. Folate deficiency was detected in 0·9 % of women, while 7·0 % had supraphysiological levels of the vitamin. No significant effects of age, educational level, marital status, parity, smoking status or nutritional status on serum folate levels were detected by univariate or multivariate analyses. Intake of folic acid supplements showed a significant association with serum folate levels, but only 1·2 % of women used supplements.The mean, median and range of serum folate were 14·2 (Folate deficiency in women of reproductive age living in the MR of Chile is almost inexistent according to the ENS 2016–2017, suggesting that the current population-wide mandatory folic acid fortification of flour is an effective and equitable measure to prevent folate deficiency. These results support the option of maintaining current folic acid fortification in Chile, particularly based on the low adherence to supplementation regimes evidenced in other populations. NTD are a group of congenital malformations resulting from incomplete closure of the neural tube, the embryonic precursor of the brain and spinal cord. Because human neural tube closure takes place 3 weeks after conception, before most women are even aware of their pregnancy, adequate folate levels should be provided to all fertile women to be effective at preventing NTD. This situation is particularly relevant in regions such as Latin America, where 69 % of pregnancies are unintended.In 1991, the UK Medical Research Council demonstrated that periconceptional supplementation with folic acid could significantly reduce the incidence of neural tube defects (NTD). In most populations, folic acid supplement use is also ineffective because of low awareness of patients and healthcare providers to the use of folic acid before pregnancy,7, together with low adherence to the use of pills and with the existence of socio-economic disparities (high costs and low availability) of vitamins in specific locations,9. Folic acid mandatory fortification of food is considered an effective measure to provide sufficient folate to women before they get pregnant. Enrichment of cereal grain products with folic acid was first implemented in the USA in 1998. Since then, folic acid fortification of wheat, maize and/or rice flour has been adopted as a cost-effective public health policy in more than eighty countries,11, and its implementation has proven to reduce the incidence of NTD significantly,13.Since the early 1990s, different strategies to increase blood folate concentration in women of reproductive age have been considered. Interventions designed to increase the intake of folate-rich foods were rapidly discarded, as they were ineffective in improving folate statuset al. showed that serum folate levels had risen from 4·3 to 16·4 ng/ml in a cohort of 605 young women from low socio-economic households. The policy showed to be effective in decreasing the incidence of NTD by 43 % from pre-fortification (1999–2000) to post-fortification (2001–2002). The cost-effectiveness of this intervention was later demonstrated by Llanos et al., who showed that the overall cost of surgery and long-term rehabilitation for patients with NTD ‘significantly exceeded the investment incurred by the milling industry’.In Chile, mandatory fortification of wheat flour with 2·2 mg folic acid/kg was first implemented in 2000. This policy was designed to increase the average intake of folic acid by 400 µg/d in women of reproductive age. In 2003, Hertrampf –21. Positive associations between high serum folate with anaemia and enhanced cognitive decline in the elderly with vitamin B12 deficiency, as well as increased risk or progression of some types of cancer, were shown both internationally and in Chile–24. High folic acid intake was also shown to interfere with the action of certain drugs, for example, those used to treat epilepsy, some autoimmune diseases and malaria. Those potential harmful effects of high folic acid intake, together with international concerns about the increased supply of foods fortified with folic acid, led the Chilean government to adjust the fortification policy in 2009 to 1·8 mg/kg wheat flour (range 1·0–2·6 mg/kg).Evidence worldwide and from Chile suggested that specific population subgroups or individuals might suffer adverse effects if exposed to high levels of folic acidIn this work, we analysed the current folate status of a subgroup of Chilean women of childbearing age for the first time after the implementation of the policy adjustment. The current study, aimed at providing evidence to assess the need for adjustment of the public policy of flour fortification with folic acid, was part of the 2016–2017 National Health Survey .All women of childbearing age participating in the ENS 2016–2017 and living in the Metropolitan Region (MR) were selected for folate determinations, because of budget and logistic constraints (further information in the last paragraph of this section). The MR is one of the sixteen administrative and political regions from Chile. Although it is one of the smallest regions, it is the most densely populated and contains the capital city (Santiago) and its urban suburbs.. The number of selected households was 9303: 23·2 % were not present at home (not reached) or were non-eligible  and 9·8 % refused to participate. The household national survey response rate (participants interviewed/participants sampled) was 67 %. No replacements were performed. The participants included in the ENS 2016–2017 survey were 6233 non-institutionalised men and women aged 15 years or more  and associated risk factors by sex and age groups. The Kish method was used to select one participant per household randomlyData were collected in two visits, lasting nearly 1 h each. During the first home visit, a lay interviewer applied health questionnaires using electronic data capture devices. Eighty-nine percentage of the interviewed participants received a second visit by a trained nurse, who applied questionnaires, measured blood pressure, conducted anthropometric measurements and collected fasting blood and urine samples. The nurse also recorded the medications participants were currently using (prescribed or not) via a detailed inventory. Medications were classified using the anatomical therapeutic chemical (ATC) classification system, in which supplements of folate and derivatives are given the code ATC B03BB . Simila,29. The maximum period of stability of folate samples protected from light, if refrigerated, is 2 d. Among 241 women in childbearing age in the MR, 225 blood samples were processed to obtain serum within 1 h of their collection and transported in <2 d at 4°C to the Clinical Laboratory Red Salud UC CHRISTUS from Pontificia Universidad Católica de Chile in Santiago. Women subjected to serum folate analyses from the MR were mainly from urban areas (n 222), and only three women lived in rural areas, so the latter were excluded from the analyses.Weight assessment was done with the HN289 OMRON digital scale ). Height was measured with a set square and a chrome-plated tape measure against a wall, as reported previouslySerum folate concentrations were measured by electrochemiluminescence immunoassay, using the Roche Diagnostics ‘Cobas 8000-E602 Immunoassay Analyzer’. The assay coefficients of variation were 10·6 and 7·3 % at mean iodine concentrations of 2·5 and 6·7 ng/ml, respectively.. The supraphysiological category was subdivided into two sub-categories: high (>20–26 ng/ml) and very high (>26 ng/ml), according to the reported cut-offs at which free folic acid is detected and cognitive deficits are reported, respectively,32.The serum folate levels were classified into deficient (<4·4 ng/ml), normal (4·41–20 ng/ml) or supraphysiological (>20 ng/ml) according to the WHO’s guidelinesv. medium, 8–12 years v. high, 13 or more years of schooling), marital status (married or cohabiting v. single or divorced or widowed), parity (number of births), smoking status (smoker v. non-smoker) and nutritional status (defined as underweight (BMI < 18·5 kg/m2), normal (BMI 18·5 to <25 kg/m2), overweight (BMI 25 to <30 kg/m2) and obese (BMI ≥ 30 kg/m2)),34. BMI (kg/m2) was derived from height and weight.The following variables were considered in the analysis: age, educational level . Associations between serum folate levels and all variables included in the study  were established by univariate and multivariate linear regression analyses, using SPSS software (version 17.0). The multivariate linear regression model for serum folate levels was adjusted for age (years) and nutritional status (BMI (kg/m2) (continuous); educational level, marital status and parity ; physical activity during leisure, current smoking status and folic acid supplementation (dichotomic). Median and percentiles were calculated using SAS 9.4 (2013) statistical software. All analyses were adjusted for the complex survey design of the ENS.Analyses were based on complete cases. 2). Only 1·2 % of the participants were taking folic acid supplements.The socio-demographic characteristics of the weighted sample are shown in Table Results indicating the prevalence of folate deficiency and excess are shown in Table P = 0·028), where women with supplementation had, on average, 70 % higher levels of serum folate than women without folic acid supplementation .The mean serum folate level of women of childbearing age living in the urban MR was 14·2 ± 0·4 ng/ml, and the median was 13·5 ng/ml (range 2·1–32·2 ng/ml). Table Linear regression was performed to examine the relationship between serum folate concentrations with age, educational level, marital status, parity, smoking status, nutritional status and folic acid supplement use Table . The onl and concomitantly reduced the incidence of NTD by 43 %. The folate status of women of reproductive age in Chile was last analysed a few years after, in 2003, in women of low socio-economic households.In 2000, the implementation of mandatory folic acid fortification of wheat flour in Chile increased circulating folate levels in women of childbearing ageThe aim of this paper was to determine the current folate status in Chilean women of reproductive age belonging to different socio-demographic groups for the first time after the last adjustment to the folic acid fortification policy in 2009. The results show that folate levels were mostly adequate in women included in the current study. This information, together with current data on NTD prevalence in Chile, suggests that the current folic acid fortification policy is effective and equitable.. Serum folate levels were adequate in 89·1 % of women, and no disparities were detected according to socio-demographic variables previously shown to affect folate status, such as educational level, smoking status or BMI. As expected, folic acid supplement intake was associated with higher serum folate levels, but only 1·2 % of the population took supplements. Despite this low level of supplement use, 7 % of women (including supplement users and non-users) exhibited supraphysiological serum folate levels.The mean serum folate concentration in Chilean women of reproductive age from the MR of Chile was 14·2 ng/ml, lower than the 16·3 ng/ml determined in Chilean women in 2003. This difference may explain the lower level in serum folate in 2016–2017 v. 2009–2010 surveys. Future evaluation of folate status in Chileans will require the analysis of the target population (young women) in parallel to other populations potentially affected by the fortification policy, for example, children and elderly.Folate concentrations detected by the ENS 2016–2017 were lower to those in the ENS 2009–2010: the mean shifted from 21·2 to 14·2 ng/ml, and the p95 from 38·6 to 24·1 ng/ml. However, a direct comparison between serum folate levels from both surveys is inadequate because different laboratory techniques were used and different subpopulations were included in the ENS 2009–2010 and ENS 2016–2017. The former used direct competitive chemiluminescence immunoassays and included elderly participants, and the latter used competitive electrochemiluminescence-based immunoassay and analysed women of reproductive age, respectively. Indeed, existing evidence shows that higher levels of serum folate are usually detected in participants older than 60 years due to higher intake and/or lower catabolism of folic acid. However, this method requires standardisation and adjustment of threshold levels to obtain reproducible and comparable results within and among laboratories. In our country, the microbiological assay method was not available when the ENS 2016–2017 was undertaken, so we used electrochemiluminescence immunoassay, a commercial protein-based immunoassay used in clinical and research settings, to determine serum folate levels.The WHO recommends the microbiological assay as the most reliable method to obtain comparable estimates of population folate status across different countries. The prevalence of NTD in 2018 was 6·4: 10 000 live births . These proportions are in the range of 4–9:10 000 live births expected in countries that have reached adequate folate status,39.The current study shows that serum folate levels are mostly within the normal range in young women living in urban areas of the MR of Chile and suggests that the current folic acid fortification policy is adequate to keep NTD within folate-preventable ranges. In a recent publication, the mean reported NTD prevalence in Chile between 2001 and 2013 was 5·5:10 000. Secondly, the current study only measured the folate levels in women of childbearing age living in the urban MR of Chile because folate integrity could not be ensured for samples transported to the clinical laboratory from other regions. Hence, our study is only representative of women residing in the MR, not in Chile as a whole. Nevertheless, it is important to note that approximately 40 % of Chileans live in the MR. Thirdly, we measured serum folate instead of erythrocyte folate, the latter being a better indicator of long-term folate levels. Finally, folate intake was not assessed in the ENS by means of dietary questionnaires . This would have enabled us to verify if women of childbearing age are meeting folate intake recommendations. Furthermore, the correlation between serum biomarker levels and women’s reported intake might have served as an indirect measure of flour manufacturers’ compliance with regulations regarding folic acid fortification in Chile.Our study has several limitations. Firstly, we were unable to determine folate insufficiency on the basis of an increased risk of NTD because of a mismatch between the assay used to establish this folate insufficiency cut-off  and the assay we used to determine folate levels in our study (electrochemiluminescence immunoassay)v. excess of folate may also be required to assess the benefit v. risk of folic acid fortification in our country objectively. Also, it is fundamental to ensure strict surveillance of the flour and food industries to prevent excessive fortification and addition with folic acid.Our analysis highlights the importance of performing additional research in order to monitor folate status in different age and sex population subgroups at a national, using the gold standard methods for measuring long-term folate status , before considering any further adjustments to the national policy. Technical calculations of the burden of disease due to deficit ,9,39,40 and the high proportion of unplanned pregnancies in our region further support this view.Altogether, the results and discussion described above support maintaining the current fortification policy with folic acid as a measure to maintain folate-preventable ranges of NTD in Chile. The low awareness/adherence and socio-economic disparities in the use of vitamin supplements


In this study, the occurrence of pesticide residues and their metabolites in grapes and wines was investigated. A targeted analysis of 406 pesticide residues in 49 wine and grape samples from organic and conventional production were performed using the QuEChERS  extraction method, followed by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry. Multiple residues (>4 residues/sample) were detected in 22 tested samples. The most commonly detected residues were fungicides  and insecticides . An ultra-high-performance liquid chromatography–high resolution mass spectrometry method (UHPLC–(HR)MS) was used for screening of pesticide metabolites. We also provide a method and database for detecting pesticide metabolites (extending our previously published database to 49 metabolites originating from 25 pesticides). An introduced strategy of targeted screening of pesticide metabolites was applied for authentication of 27 organic grapes and wines. In total, 23 samples were free of quantifiable residues/detected metabolites or contained residues approved for organic production. Botrytis cinerea), downy mildew (Plasmopara viticola), powdery mildew (Uncinula necator), and grape moth (Lobesia botrana). In conventional production, various plant protection products (PPP), especially fungicides and insecticides, are widely used for protecting grapevine. For example, pesticide preparations with active ingredients such as fenhexamid, boscalid, dimethomorph, iprovalicarb, penconazole (fungicides) or methoxyfenozide, imidacloprid (insecticides) are approved in the European Union and commonly used by growers [Grapes are among the most widely grown fruits worldwide, consumed both fresh and in the processed forms . Approximately 50% of grapes are used in wine production . The vin growers ,3,4. AltThe data in The 2018 European Union report on pesticide residues in food  clearly Under these conditions, it is not surprisingly that consumers’ demand for organic grapes and wines is continuously growing. Organic farming is aimed at avoiding the use of artificial pesticides and fertilizers, thus reducing possible impact on the environment. Also, consumers’ dietary exposure to pesticide residues in organic food is fairly lowered compared to consumption of products from conventional farming in which the application of various pesticide preparations against the harmful organisms is a common agriculture practice. The plant protection products approved in organic production are listed in the Commission Regulation (EC) No. 889/2008 (Annex II) ,8,9. GenIn most available studies, a two-step sample preparation procedure is usually used for extracting pesticide metabolites from grape samples. In the first step, metabolites are extracted using either a polar solvent, such as acetonitrile, methanol or their mixture with water; alternatively, the “Quick, Easy, Cheap, Effective, Rugged and Safe” (QuEChERS) method or liquid–solid extraction are widely used for isolation of pesticide metabolites from grape samples. In the second step, a clean-up by dispersive solid phase extraction is often performed, using sorbents such as primary secondary amines (PSA), octadecyl sorbent C18) or graphitized carbon black (GCB) ,15,16,17 or graphIn our previous study , the strTM or Honeywell Riedel-de HaenTM . The purity of standards ranged from 90 to 99.9%. Triphenyl phosphate (TPP) and nicarbazin , HPLC-grade acetonitrile, LC–MS grade formic acid, ammonium formate and ammonium acetate were supplied by Sigma-Aldrich . Methanol was obtained from Merck . Acetone p.a. and sodium chloride were purchased from Penta . Anhydrous magnesium sulphate was obtained from Honeywell FlukaTM. Deionized water (18 MΩ) was produced using a Millipore Milli-Q system .Certified standards of pesticides were obtained from Dr. Ehrenstorfer GmbH , Honeywell Flukav/v) (depending on the solubility of the pesticide standard). A composite standard solution in acetonitrile was prepared at 50,000 ng/mL (each) from individual stock solutions. The working standard mixtures (20–2000 ng/mL) used for matrix-matched calibration were prepared from stock solution by diluting with acetonitrile. All standard solutions were stored in a freezer at −18 °C. Certified standards of pesticide metabolites were not commercially available [Individual pesticides’ stock solutions and internal standards were prepared in pure acetonitrile, methanol or acetone containing 1% formic acid  for 12 h and then homogenized using a laboratory blender. The overview of samples is shown in 4) was added and the shaking process was repeated. Then, an internal standards mixture was added and the tubes were centrifuged. An aliquot of the supernatant was transferred into a vial for LC–MS analysis.The procedure for isolation of parent pesticides and their metabolites was similar (QuEChERS method) as described in our previous study . BrieflyThe analyses of 406 pesticide residues were performed using the ultra-high-performance liquid chromatograph Waters Acquity UPLC system coupled to a triple quadrupole tandem mass spectrometer Xevo TQ-S  in electrospray positive (ESI+) and negative (ESI-) modes. Sample separation was performed using an Acquity UPLC HSS T3 analytical column .v/v) formic acid and (B) methanol, respectively. For compounds detected in the ESI-, mobile phases were (A) water with 5 mM ammonium acetate and (B) pure methanol. The gradient was the same in both polarities: the starting mobile phase composition was 10% of the organic phase (B) with a flow rate of 0.3 mL∙min−1, which was linearly changed over 1 min to 50% (B), along with the linear change of the flow rate to 0.35 mL∙min−1. Then, the mobile phase was linearly changed from 50% to 95% (B) over the next 9 min, along with a gradual increase of the flow rate from 0.35 to 0.45 mL∙min−1. Over the next 1 min, the mobile phase changed to 100% (B) and the flow rate increased from 0.45 to 0.6 mL∙min−1; this composition was held for the next 2 min. The column was subsequently reconditioned for 2 min in the starting mobile phase composition, i.e., 10% (B), at a flow rate of 0.5 mL∙min−1. Sample volumes injected in the positive and negative ESI modes were 2.5 μL and 3 μL, respectively. The column and autosampler temperature were maintained at 40 °C and 5 °C, respectively. For compounds detected in the ESI+, mobile phases consisted of (A) water with 5 mM ammonium formate and 0.1%  was operated in the multiple reaction monitoring (MRM) mode. Electrospray ionization was conducted in the positive and negative mode with the capillary voltages of 0.6 kV and −0.6 kV, respectively. The source and desolvation temperatures were 120 °C and 350 °C, respectively. Nitrogen was used as the desolvation and cone gas, argon was used as the collision gas. The generated data were processed by MassLynx software .The ultra-high-performance liquid chromatograph Agilent Infinity 1290 LC system coupled to Quadrupole-Time of Flight high resolution mass spectrometer (UHPLC–HRMS) Agilent Ion-Mobility Q-TOF 6560  in positive and negative ESI modes was used for the analyses of pesticide metabolites. Sample separation was performed using an Acquity UPLC HSS T3 analytical column . The parameters of the UHPLC–HRMS analysis were described in detail in the previous study .Pesticide residues were identified on the basis of a combination of the retention time and detection of two MRM transitions (considering their ion ratio); those were previously acquired from pesticide reference standards. The identification criteria were in accordance with the requirements stated in the European Commission’s guidance document SANTE/12682/2019 .The strategy of detection and identification of pesticide metabolites was based on the calculated accurate mass, isotopic pattern matching and the accurate mass of MS/MS fragments. The acceptable mass error of potential elemental composition for quasi-molecular ions was ±5 ppm. A detailed description can be found in the previous study .The analytical method for analysis of pesticide residues in grapes and wines was validated. Recoveries, repeatabilities and limits of quantification were determined in accordance with the European Commission’s guidance document “Analytical quality control and method validation procedures for pesticide residues analysis in food and feed” . ValidatThe analytical method for pesticide residue analysis is accredited and routinely performed according to the EN ISO/IEC 17025:2017 standard. The laboratory regularly participates in the official European Union Proficiency Testing Program to control and keep the quality and accuracy of results.The presented method was validated for 406 pesticide residues in grapes and wines according to the European Commission’s guidance document “Analytical quality control and method validation procedures for pesticide residues analysis in food and feed” . Limits In this study, 10 grape and 12 wine samples from conventional production were analyzed. All tested samples contained quantifiable residues of more than one pesticide see . In totaow) and technology of the wine-making process  [The transfer of pesticide residues from grapes to wines, expressed as processing factor , was influenced by the physicochemical properties of residue  . As no lduction) . Howeverduction) ,26,27. OThe full results of pesticide residue analysis in tested grapes and wines are detailed in the In our previous study, the database of 18 metabolites originating from 7 pesticides was established . Here, wSince standards of metabolites were not commercially available, determination of metabolite concentrations in tested samples could not be performed. Therefore, the results were presented as “relative response”—the ratio between the area of the detected metabolite and the area of the internal standard .Pesticide metabolites were detected in all tested samples. Altogether, 41 different pesticide metabolites originating from 22 pesticides were found. The metabolite of fenhexamid, fenhexamid-hydroxy, was the most commonly detected biotransformation product in both grapes and wines. Metabolites of iprovalicarb, penconazole, pyrimethanil, cyprodinil and metalaxyl were often found see , too. FoThe full results of the targeted screening of pesticide metabolites in tested grapes and wines are summarized in the The strategy based on combined targeted screening of pesticide residues and their metabolites was applied for authentication of organic products. In this study, 6 organic grape samples and 21 organic wine samples were analyzed for 406 pesticide residues and 49 pesticide metabolites. In total, 23 out of 27 samples were free of quantifiable residues/detected metabolites or contained residues linked to agricultural practices permitted in organic production see . HoweverConsidering available PFs for wine production , the levThis study presents the results of the analyses of pesticide residues and metabolites in grapes and wines from both conventional and organic production. Fungicides and insecticides were the most commonly detected pesticide residues; in total, residues of 29 and 25 different pesticides residues were found in grapes and wines, respectively. Screening of pesticide metabolites was performed using the recently extended database of pesticide metabolites containing 49 compounds . Altogether, 41 different pesticide metabolites originating from 22 pesticides were detected in tested samples. The follow-up research should focus not only on further database extension, but also metabolites quantification in order to estimate the extent of parent compound transformation, hence its original concentration. Several samples contained no quantifiable residues of parent pesticides (or with their residues below the quantification limit of 0.01 mg/kg), while metabolites of such pesticides were unequivocally found. This was observed in the case of cyprodinil, dimethomorph, fenhexamid, fluopyram, mepanipyrim and penconazole in grapes, and benalaxyl, fenhexamid, iprovalicarb, metalaxyl and pyrimethanil in wines. The control strategy of analyzing pesticide metabolites as markers of unauthorized practices in organic farming was applied to a set of organic samples. In total, 15% of tested organic samples contained quantifiable residues or detected metabolites. In the latter case (no parent residues detected), such samples would pass a routine residues control without the doubts on their origin when declared as “organic”. Our results indicate that the combined analysis of parent pesticides and their metabolites represents a promising tool for tracing history of pesticide application on various crops; moreover, it enables obtaining the evidence on an unauthorized application of plant protection products in organic production.


Watersipora subatra seemed to be inhibited by the biofilm developed in the MPA, Diplosoma cf. listerianum seemed to be attracted by biofilm developed in the MPA only under mid anthropogenic pressure. The obtained information is critical for marine environmental management, urgently needed for the establishment of prevention and control mechanisms to minimize the settlement of NIS and mitigate their threats.Microbial biofilms can be key mediators for settlement of macrofoulers. The present study examines the coupled effects of microbial biofilms and local environmental conditions on the composition, structure and functioning of macrofouling assemblages. Settlement of invertebrates over a gradient of human-impacted sites was investigated on local biofilms and on biofilms developed in marine protected areas (MPAs). Special attention was given to the presence of non-indigenous species (NIS), a global problem that can cause important impacts on local assemblages. In general, the formation of macrofouling assemblages was influenced by the identity of the biofilm. However, these relationships varied across levels of anthropogenic pressure, possibly influenced by environmental conditions and the propagule pressure locally available. While the NIS  Biofouling is the undesirable attachment and growth of marine organisms on submerged artificial structures that leads to economic, ecological and safety-related negative effects ,2,3. ForIn aquatic environments, biofouling on hard substrates starts as a conditioning film, which leads to biofilms and, subsequently, to complex mature macrofouling communities ,11,12. MPhallusia nigra, the serpulid Pomatoceros lamarkii, the barnacle Semibalanus balanoides or several mussels [Balanus trigonus [Spirorbis  amphitrite, which avoid young biofilms but are induced to settle on older biofilms with more diverse microbial composition and abundance [Hydroides elegans . Alt. AltPhals  the settlement of native and NIS macrofouling species will be modulated by the presence of particular microbial biofilms; and b) the local environmental conditions will influence both micro and macrofoulers. For that, we investigated how biofilms developed in pristine and impacted habitats affected the settlement of invertebrate larvae over a gradient of human-impacted sites. Additionally, both biodiversity and functioning of macrofouling assemblages were investigated. This information is urgently needed for future optimization of standardized surveys and the establishment of prevention and control mechanisms to mitigate threats posed by NIS.p < 0.01). While cyanobacteria were absent in almost all pressure levels, green algae reached mean values of approximately 0.014 µg cm–2 in sites exposed to high and low anthropogenic pressures, and up to mean values of 0.034 µg cm–2 under mid pressure. On the contrary, diatoms reached the maximum values in sites submitted to low anthropogenic pressure, while minimum values were measured under mid and high pressures and in the marine protected area (MPA) (see –2 in sites submitted to low anthropogenic pressure (see p < 0.01), but the lower dispersion of replicates  did not provide a significant contribution to differences between pressures  changed significantly with “Pressure”, “Site” and “Panel” a, althouMPA) see . After 3sure see . In thisressures b.Francisella and Pseudofulvibacter, being both more abundant under high pressure. Thiothrix showed an opposite trend, with greater abundances under low compared to high and mid pressures and even on MPA. Francisella also contributed to the dissimilarity between high vs. mid and vs. MPA, as well as Halocynthiibacter, which was abundant under high pressure and absent under mid pressure and on MPA. Alcanivorax and Pseudofulvibacter showed greater abundance under mid vs. low pressure, while Truepera showed an opposite tendency, also contributing to the dissimilarity between low vs. MPA, where it was absent. The genus Proteobacteria bacterium JGI 0000113-L05 and bacterium WHC4–2, contributed to dissimilarity between high vs. MPA, the later also showing larger abundance under high compared to mid pressure. The genera Verticia and Marivita showed large abundance under mid pressure and were absent at MPA, while unidentified marine metagenome and uncultured bacterium and prokaryote showed larger abundances at MPA (detailed in Mid > 283.5Low = 257MPA = 236High).Biofilm sampled after 30 days of panel deployment revealed that the structure of the bacterial assemblages at the genus level varied significantly depending on anthropogenic “Pressure” c, althouGrammatophora oceanica showed greater cover under low anthropogenic pressure than under high or mid pressures, while Bacillaria socialis showed the inverse trend. As for B. socialis, Licmophora flabellata contributed to the dissimilarity between low vs. mid anthropogenic pressures, being scarcer under low pressure. Licmophora flabellata and Nitzschia aff. distans were the diatoms contributing the most to the dissimilarity between mid and high pressure. While the first was abundant under mid pressure, the latter only appeared in the highly impacted sites. Taxa such as Achnanthes aff. delicatissima or Parlibellus aff. coxieae showed great cover in MPA compared to high pressure, where both species were absent.Due to insufficient material collected in Moaña (mid pressure site), only six sites were used for diatom analysis. A total of 113 diatom taxa were identified; 25 taxa registering abundance over 3% in at least one sample. The structure of diatom assemblages varied significantly across levels of anthropogenic “Pressure” , althougA total of 75 taxa were identified across all study sites. Eight were classified as non-indigenous species (NIS), 14 as native to the area, 6 as cryptogenic and 47 as unknown. While the native category  showed a total mean cover of 68.5%, NIS reached a total mean cover of 5.4%.p < 0.001), with the maximum and the lowest values of cover at mid and low pressure, respectively  and low pressure   x Or”, eo site) .p < 0.01) and mid versus low pressure (p < 0.05)  b. Simila < 0.05) c,d, and The present study fully supports our working hypothesis that the settlement of macrofouling species, involving both native and non-indigenous species, was modulated by the coupled effects of composition and structure of microbial biofilms and the local environmental conditions where they develop, demonstrating their relevance in determining the structure and functioning of resulting macrofouling assemblages.a were already detected during the first days of the study, as previously reported in other regions . Add. Adda wes  for macrofouling recruitment. Many environmental variables can affect the successional patterns of the colonization on hard substrates, and salinity, temperature or water pollution exert obvious influences on the structure of biological assemblages and, consequently, on their functioning e.g., ,41,42). ,42. 41,4Recruited macrofouling assemblages, as well as their diversity and evenness indices, were also influenced by the identity of the biofilm, with lower diversity and evenness values detected in assemblages settled on biofilm developed at the MPA. Some studies already found that the settlement of larvae was strongly affected by locally-developed films at a particular site, also suggesting that the density of the microbial film could determine the attractiveness to settling larvae, and that these effects could not be easily decoupled . MoreoveWatersipora subatra) was suppressed by the presence of an MPA biofilm, suggesting that characteristics of the biofilms, as chemical cues or parameters related with community structure or extracellular products, could affect its activity, as already shown by [Mytilus galloprovincialis. This supports the capacity of biofilms as key players in determining the colonization process of macrofoulers and, consequently, on the structure and functioning of biological assemblages [Wahl et al.  already shown by  for Mytiemblages .Spirobranchus triqueter appearing settled mostly on MPA biofilms and the NIS Watersipora subatra on local biofilms.Increasing the propagule pressure of NIS, because of marine traffic pressure, is an important factor in controlling the establishment and stability of non-native populations worldwide . We founTricellaria inopinata and Watersipora subatra. In particular, our study detected the capacity of biofilm organisms from pristine habitats in modulating the settlement of particular NIS . However, in view of the observed tendencies across anthropogenic pressure or origin of biofilm , we suggest a combined effect of the initial composition of the biofilm and the environmental conditions  in modulating the macrofouling assemblages on marine environments. As previously stated, biofilm can have an important role in larval and spore settlement of organisms , but thels  have long been considered to be key mediators for the settlement and the subsequent colonization by macro-organisms in many studies ,22,23,24Several sites distributed across the coast of Ría de Vigo (NW Spain) and exposed to different environmental conditions and anthropogenic pressures (see 43–46), between 2 and 15 km apart, were selected. These sites were grouped into the following four categories: i) a floating pier located within a marine protected area (MPA), i.e., recruiting biofilm assemblage to a stage close to pristine; ii) small harbors without environmental protection, but with almost no maritime traffic, i.e., low pressure; iii) ports affected by medium anthropogenic pressure, including short ferry crossings, aquaculture activities and recreational boats, i.e., mid pressure; and iv) ports affected by high anthropogenic pressures, including fisheries, recreational vessels and commercial traffic, i.e., high pressure. Two suitable experimental sites were selected in each of the selected anthropogenic pressure level categories with the exception of the MPA, where only one marina was available .3) were individually attached to a brick from buoys or docks between 1 and 2 m depth. Panels were suspended horizontally facing downwards and separated at least 1.5 m within each site. Only the sides of PVC panels facing down were used for measurement and sampling, a standard methodology mimicking floating docks widely used for the study of fouling macroinvertebrates communities [Based on the design employed by , on 7–8 munities . Six pan–2) on the side of PVC plates facing down was non-destructively examined 15 and 30 days after the deployment by using a BenthoTorch . This spectrofluorometric tool enables the monitoring and characterization of phytobenthic assemblages directly in the field and is especially indicated for the quantification of biofilm biomass at the early stages of their development [a and diatom concentrations (n = 3 per panel).The weight of biofilms (µg cmelopment . RecordeOne month after the start of the experiment (4–5 February 2019), which was considered enough time to obtain a mature biofilm  according to the manual. DNA was re-suspended in a final volume of 100 µL. An extraction blank was included in every DNA extraction round and treated as a regular sample to check for cross-contamination. For library preparation, a fragment of the bacterial 16S rRNA region of around 450 bp was amplified using the universal primers 341F R (5’ CCT ACG GGN GGCW GCA G 3’) and 805R (5’ GAC TAC HVG GGT ATC TAA TCC 3’) [ TCC 3’) . The seq TCC 3’) ; QIIME r TCC 3’) . OperatiDiatoms. For analysis of diatom assemblages, the samples were cleaned with 4–6 mL of 65% HNO3 and K2Cr2O7 at room temperature for 24–48 h. Afterwards the samples were repeatedly centrifuged (1500 rpm) and rinsed with distilled water to remove oxidation by-products. Finally, permanent slides were mounted using Naphrax® (UK). Two slides (22 × 22 mm2) of each sample were examined for diatom content using a light microscope  and a 100× immersion objective (NA 1.25). Diatoms were identified to the lowest taxonomical level possible by using a specialized bibliography [iography ,84,85,86The extra panels deployed at the MPA colonized by pristine biofilms were transplanted to the sites exposed to different anthropogenic pressure , all panels were retrieved from the field and taken to the laboratory to assess the composition and functioning of macrofouling assemblages. The whole surface of each panel was photographed with an Olympus TG-4 camera . Panels of each site were maintained separately in 50 L tanks, in an open seawater system with 10 µm filtered UV irradiated seawater at 16–18 °C in an acclimated room (18 °C).All sessile macroinvertebrates and macroalgae were identified within the following 2 days after collection to the lowest possible taxonomic level with the aid of a stereomicroscope  and specialized bibliography and then assigned to four categories as follows: native; NIS; cryptogenic  in accordance with the literature . Conseq–1; Eheim, type 1001.220, GmbH and Co KG). Variations in oxygen concentration were measured every 30 s using a luminescent dissolved oxygen probe connected to a data-logger . Respiration (measured as mg O2 h–1) was estimated by regressing oxygen concentration in the chamber over time, with the caution of monitoring chambers without assemblages to correct for potential bacteria and phytoplankton respiration rates. Estimates were also corrected by volume of seawater inside the chamber taking into account the different volumes of assemblages. Water inside the chambers was renewed every new incubation.We considered the biomass (the wet weight (g), obtained after subtracting the biomass of an empty wet PVC panel from the weight of the colonized one) and respiration as ecosystem functioning surrogates. Respiration rates were estimated by measuring oxygen fluxes during dark periods using 4 L acrylic incubation chambers sealed and submersed in the tanks. Water movement was maintained in the incubation chamber using a submersible pump  based on untransformed data and Bray–Curtis dissimilarities. The analysis included pressure , site  and panel  as factors (n = 3). PERMANOVA was also conducted on a square-root transformed percentage of diatoms and on a fourth-root transformed number of OTUs of bacteria observed at the genera level using Bray−Curtis similarities, but in this case including “Pressure” as the only fixed factor. Pair-wise comparisons were done after obtaining a significant factor or a significant interaction of factors. When the number of unique permutations for the pair-wise comparison was less than 30, Monte Carlo p-values (P(MC)) were considered [Changes in the weight of biofilm in panels, i.e., µg cmnsidered . A test nsidered . Non-metnsidered , and to For each plate, relevant descriptors of community structure such as species richness, Shannon diversity and Pielou evenness indices were calculated. Since many of the organisms could not be identified to species level, we used the term taxonomic richness rather than species richness. Univariate data such as total percentage cover of macrofoulers and the most conspicuous NIS, taxonomic richness, Shannon index and Pielou evenness, biomass and respiration rates were analyzed using PERMANOVA based on Euclidean distances. The model included “Pressure” and “Site” (as previously described) and “Origin of biofilm”  as factors. These analyses were also performed considering separately total percentage cover and taxonomic richness of NIS species.Changes in macrofouling assemblages were examined using PERMANOVA multivariate procedures based on the square root-transformed data and Bray–Curtis dissimilarities following the same previous model. Pair-wise comparisons were performed when significant differences were found. PERMDISP, nMDS and SIMPER analyses were also carried out.The software PRIMER v6 and PERMANOVA+ for PRIMER  were used for all data analyses ,100, and


Previous studies showed that adding zero valent iron (ZVI) can increase the methane production and degradation rate of organic waste by improving the performance of anaerobic digester. However, our study firstly found that ZVI  inhibited the anaerobic digestion (AD) of cow manure and lignocellulose. ZVI significantly increased the methanogenic rate of cow manure in the first 6 days, but decreased the accumulative methane yield and volatile fatty acids yield by 10.3 and 12%, respectively. The effect of ZVI on AD of liquid biomass separated from cow manure was positive, but the effect on solid biomass was negative. These results indicated that ZVI enhanced the AD of easily biodegradable organics but inhibited the biodegradation of refractory organics (lignocellulose). By analyzing the varying effects of ZVI in diverse anaerobic systems, it was found that the effects were influenced by the characteristics of substrate and inoculum-substrate ratio. This study suggested that only proper ZVI addition can improve the AD process depending on the feeding materials. Anaerobic digestion (AD), with the merits of reducing organic pollution, energy recovery, and low operation cost, has become a widespread pathway in converting organic waste to energy –(3) —a strong reductive material—is among the most prominent additive to enhance the AD of wastewater and waste activated sludge due to its non-toxicity, abundance, low cost, and easy manufacturing  and cow manure under varying inoculum-substrate ratio (ISR). ZVI played diverse roles in different AD systems depending on the components of substrate, which was studied and summarized to provide a reference for the practical application of ZVI in AD system.Cow manure and untreated inoculum were collected from the same dairy farm of Beijing, China. Waste activated sludge was collected from a municipal wastewater treatment plant of Beijing, China. The cow manure was stored in a sealed bag and placed in a refrigerator at −20°C before experiments. The inoculum was incubated with glucose for 2 weeks, and removed the supernatant before use. Waste activated sludge was stored in a serum bottle and placed in a refrigerator at 4°C before experiments. Microcrystalline cellulose (90 μm) and starch (AR) were purchased from Shandong Xiya Reagent Co., Ltd. and Sinopharm Chemical Reagent Co., Ltd., respectively. ZVI  was purchased from Aladdin Reagent Co., Ltd., China.Cow manure was washed with an equal weight of deionized water and extruded by a sieve (0.2 mm) for solid–liquid separation. The separated liquid was labeled as liquid cow manure, and the separated solid was labeled as residue. The main characteristics of cow manure, liquid cow manure and residue are listed in inoculum:VSsubstrate = 2:1) of substrate and inoculum in a 500 mL serum bottle (working volume = 400 mL) was placed in a water bath at 36 ± 1°C. All serum bottles were sealed with butyl rubber stoppers after flushing with nitrogen, and stirred for 1 min every 5 min at 120 rpm by an automatic stirring rod. A serum bottle (working volume = 80 mL) containing 3M NaOH was connected to the reactor through a natural latex tube to fix CO2. Biogas volume (without CO2) was automatically recorded after entering the gas volume measuring device through a natural latex tube.The effects of ZVI on AD of several substrates were investigated using BMP tests. BMP tests were conducted by an Automatic Methane Potential Test System II , as described below. The mixture  and a ZVI addition group (labeled as Z). Six sets of BMP tests under ISR of 2 were set up as follows: cow manure , liquid cow manure , residue , microcrystalline cellulose , starch soluble  and cow manure inoculated with untreated inoculum . To explored the effects of ZVI on the hydrolysis–acidification of cow manure, 50 mM sodium 2-bromoethanesulfonate (SBES) was added in C-CM2 and Z-CM2 to remove methanogens . Soluble polysaccharide and soluble proteins were measured by using phenol–sulfuric acid method and Bradford Protein Assay Kit, respectively, with a microplate spectrophotometer . Volatile fatty acids (VFAs) were analyzed by a gas chromatograph  equipped with a flame ionization detector. The operating temperatures of the oven, injection port, and detector were 130, 220, and 250°C, respectively. The injection volume was 1 μL, and He was the carrier gas. The composition of biogas was analyzed using the same gas chromatograph equipped with a thermal conductivity detector. The temperature of the detector was 70°C.TS and VS were determined by differential weighing after drying at 105 °C overnight and by subsequent incineration at 550°C, respectively, according to standard methods. pH was directly determined with a pH meter . Cellulose, hemicellulose, and lignin contents of cow manure and residue were determined by Van Soest. Starch content was determined by the sulfonic acid ketone method. Carbon and nitrogen contents in dried biomass materials were detected by an elemental analyzer . For determination of the total metallic iron, cow manure and residue were digested with HNOt-test analysis and p < 0.05 was considered to be statistically different.SPSS (SPSS 24.0) was used for The theoretical moles of methane for each mole of the substrate was calculated by the following stoichiometric equation  of the substrate was calculated using the theoretical moles of methane by the ideal gas law, as shown in Equation (5):The theoretical methane potential  was calculated by the accumulative methane yield  under the BMP test in comparison with its theoretical value as follows . During the AD of cow manure, VFAs concentration was always at a lower level compared with that in other studies, and remained far below the inhibitory levels, which indicated that methanogens were sufficient in both groups to convert acetic to CH4 in time, thus hydrolysis process was the rate-limiting step in AD of cow manure. ZVI had no effect on VFAs production from blackwater  and from methylated C1 compounds to a minor extent. ZVI sustained a high syntrophic hydrogenotrophic methanogenesis activity . At the end of the experimental time, the VFAs concentration in Z-CM2 was 3539.8 mg/L, which was 12.0% lower than that of C-CM2. The higher accumulative VFAs production in Z-CM2 from day 5 indicated that ZVI suppressed the hydrolysis and acidification of refractory organic substances, such as lignocellulose. Therefore, ZVI decreased VFAs production by inhibiting the hydrolysis–acidification processes of cow manure, thereby decreasing the accumulative methane production.The most crucial product in the acidification phase is VFAs, because it is converted to acetate, HAccording to the analysis of anaerobic performance in four reactors, ZVI had different effects on AD of various organics due to their varying biodegradability. Therefore, cow manure was separated to liquid part (liquid cow manure) containing easily degradable organics  and solid part (residue) containing refractory organics for BMP tests. Lignocellulose was the main refractory organic component of cow manure in this study—up to 51.61%, and difficult to be degraded due to its complex structure n, whose theoretical methane potential was 414.81 mL/g VS. According to Equation (6), the level of anaerobic biodegradability of starch was 84.19%, which is much higher the cellulose of 46.59%. Daily methane yield in Z-MC was 6.0 mL/g VS/day on the first day, whereas it was 0.9 mL/g VS/day in C-MC, demonstrating that ZVI could shorten the start-up process of AD. The comparison of accumulative methane yield in Z-MC and C-MC showed that ZVI had no effect on the anaerobic degradation of cellulose. Microcrystalline cellulose was almost completely degraded on day 9, whereas the daily methane production in Z-R was gradually lower than that of C-R from day 8 . Compared with lignin and xylose, cellulose could be degraded to produce a considerably higher accumulative methane production per gram, and contributed to most methane production in lignocellulose . As this reaction proceeded, the precipitation of Fe(OH)2 became favorable, and then Fe(OH)2 might be transformed to magnetite according to Equation (8)  methane was produced from waste activated sludge than the control during the whole period by adding ZVI at the studied six levels , which was consistent with the results of waste activated sludge treatment in other studies  iron could stimulate the activities of enzymes involved in methanogenesis (Wei et al., 4, resulting in a stable and favorable condition for microorganism and methane production increase.By contrast, the main carbon source in the AD of cow manure and residue was crop straw, which was difficult to be degraded because of its complex lignocellulosic structure. According to the experimental results of this study in This study firstly investigated the effect of ZVI on the AD of cow manure. The accumulative methane and VFAs yield decreased by 10.3 and 12% with adding 10 g/L ZVI, respectively. ZVI decreased the methane yield of residue by 20.4%, while increased that of liquid cow manure by 15.2%, and did not change that of starch and microcrystalline cellulose. Experimental results indicated that ZVI promoted the AD of easily degradable organics, such as protein, lipid, and soluble carbohydrate, but inhibited the hydrolysis–acidification process of lignocellulose by reducing the available surface area and the accessibility of cellulose. With the increasing ISR, the effect of ZVI on methane production of cow manure changed from negative to positive. Further investigation indicated that the pretreatment of lignocellulosic biomass may be required before adding ZVI to enhance hydrolysis process, and then to increase methane yield.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ZL, LZha, XZ, and SC conceptualized the study. YM, XW, and WB completed the experiment and data analysis. YM, ZL, LZhe, and XW wrote and revised the manuscript. All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


First, we used multiple linear regression to correct a large spectral dataset (707 cows from 13 farms) for fixed effects prior to multivariate statistical analysis with principal component analysis (PCA). Results showed that farm of origin accounted for up to 57% of overall spectral variation, and nearly 80% of variation for some individual metabolite concentrations. Parity and week of lactation had much smaller effects on both the spectra as a whole and individual metabolites . In order to assess the effect of fixed effects on prediction accuracy and biomarker discovery, we used orthogonal partial least squares (OPLS) regression to quantify the relationship between NMR spectra and concentrations of the current gold standard serum biomarker of energy balance, β-hydroxybutyrate (BHBA). Models constructed using data from multiple farms provided reasonably robust predictions of serum BHBA concentration (0.05 ≤ RMSE ≤ 0.18). Fixed effects influenced the results biomarker discovery; however, these impacts could be controlled using the proposed method of linear regression spectral correction.Most livestock metabolomic studies involve relatively small, homogenous populations of animals. However, livestock farming systems are non-homogenous, and large and more diverse datasets are required to ensure that biomarkers are robust. The aims of this study were therefore to (1) investigate the feasibility of using a large and diverse dataset for untargeted proton nuclear magnetic resonance ( This in calving , can the calving ,7. The aAnother aim of dairy cattle metabolomic studies is to identify biomarkers which enable early identification of health disorders in the transition period such as ketosis ,9, hypoc1H NMR-based metabolomic studies of livestock have involved relatively small numbers of animals, often of a single breed, and often located on a single farm. In their review, Goldansaz et al. [To date, most serum z et al.  identifiz et al. . Howeverz et al. , farms [z et al. , and of z et al. . Howeverz et al. ,18, thesLinear models are routinely used by quantitative geneticists to account for the influence of systematic environmental effects  known to have significant effects on phenotypic variation , and thu1H NMR serum metabolome of clinically healthy dairy cows in early lactation. We propose a method that uses linear models to correct spectra for fixed effects and demonstrate its potential utility by quantifying the relationship between 1H NMR spectra and the current gold-standard serum biomarker of energy balance, β-hydroxybutyrate (BHBA) [The aim of this study was therefore to investigate the feasibility of using of large and diverse datasets in livestock metabolomics studies by examining the effects of fixed environmental and physiological effects on the e (BHBA) ,23. Serum samples were collected from 707 early lactation cows (<30 d in milk) from 13 farms located in southeastern Australia. Descriptive statistics of the animals included in the experiment, including herd of origin, stage of lactation , parity and serum BHBA concentrations and are summarized in 1H NMR spectra were complex; however, more than 20 metabolites could be identified. Spectra were dominated by organic acids, amino acids, glucose and phospholipid intermediates . Plots of the first 2 principal components (PCs) identified several samples located outside the 95% confidence level. These spectra were manually inspected, and a single outlier with erroneous phasing was identified and removed from subsequent analyses.PCA was repeated after outlier removal. The first 13 PCs explained greater than 90% of the variation in the spectra. Scores plots of the first three PCs, which explain 47.64%, 15.59%, and 7.45% of variation, respectively, are shown in Principal component analysis (PCA) was repeated on spectra that had been corrected for (1) WIM, (2) Parity, (3) Herd, and (4) WIM, Parity and Herd simultaneously  (Models 1 to 4). Results derived from spectra corrected separately for WIM and Parity are nearly identical to uncorrected spectra . By contIn order to quantify the effect of each fixed effect on NMR spectra, we calculated Pearson’s correlations between scores for the first three PCs from the previously described PCAs . The lar1H NMR spectra, we used conditional Wald F statistics derived from multiple linear regression models on the first three PC scores (Model 5). The higher the F statistic, the greater the effect of that variable on the PC score, and the lower the P value, the greater the statistical significance. Results derived from these models are summarized in To test the statistical significance of fixed effects on 2 values obtained from models 1–4 were used to investigate which regions of the NMR spectra were most strongly influenced by the fixed effects. As the signal intensity at each chemical shift was treated as a separate response variable, the R2 values from Models 1, 2, and 3 describe the effect of WIM, parity, and herd on each of the 24,349 chemical shifts, respectively. These R2 values were color-coded, and overlaid on an average NMR spectrum. Plots showing the effects of WIM and Parity were unremarkable  were consistent with results of linear regression spectral correction and are shown in 1H NMR spectra can be used to predict serum BHBA concentration with good accuracy. This result is expected, as BHBA is directly quantifiable from NMR spectra. The overall robustness of our approach was assessed using a “leave-one-farm-out” external validation of OPLS models built using uncorrected data. This involved iteratively setting aside data from one farm, training models using data from the remaining 12 farms, then using the withheld data for external validation. R2 results were variable (0.30 ≤ R2 ≤ 0.99), however RMSE values remained relatively low (≤ 0.18) (Our results show that (≤ 0.18) . Interes1H NMR metabolomic data was determined by comparing the results of OPLS models built using (1) data from Farm 1 only (used as a control), (2) uncorrected data from all farms, and (3) data from all farms corrected for all fixed effects. Fixed effects appeared to have minimal effect on the predictive ability of models. We observed similar 10-fold cross validation prediction accuracies for all 3 datasets . Given that we also observed clustering and separation of the 12 Holstein-Friesian herds in the initial PCA , it appePre-analytical sample handling and processing have been shown to have significant effects on human metabolomic data , and conOur results suggest that stage of lactation appeared to have a minimal effect on the NMR spectra. This is consistent with the findings of Ilves et al.  who foun1H NMR serum metabolome. We could find no other reports in the literature describing the effect of parity on the entire serum metabolome. However, our results are consistent with other studies that showed parity has a significant effect on the concentration of several metabolites in serum including glucose, creatinine, urea and BHBA [Parity appeared to have a small but significant (P < 0.05) effect on the overall and BHBA ,38,39. T2 values varied significantly depending on which farm was used for validation (0.30 ≤ R2 ≤ 0.99); however, the R2 is known to be affected by the range of the dataset, and RMSE is often considered to be a better predictor of model performance [2 ≤ 0.18) were close to those obtained from 10-fold cross validation of models built using only Farm 1 data (RMSE = 0.12) and all data (RMSE = 0.10). The fact that prediction errors were highest when Farm 1 data were withheld for validation suggests that the increased variation observed in Farm 1 data represents valuable biological variation rather than confounding/noise. Despite the significant influence of fixed effects on the serum metabolome, results obtained from the leave-one-farm out external validation suggest that prediction models constructed with data from multiple farms are quite robust. Rformance . PromisiCorrecting data for fixed effects had very little impact on the predictive ability of OPLS models. Furthermore, when corrected spectra were used, y-values also had to be corrected, making interpretation of phenotypic values difficult. Interestingly, Wanichthanarak, et al.  found thLoadings from OPLS models built using uncorrected spectra from Farm 1, and spectra from all farms corrected for fixed effects, were consistent with the literature. BHBA and glucose concentrations have been shown to be negatively correlated in the serum of cows in early lactation dairy cows . L-lactaThe fact that loadings were different when uncorrected spectra from all farms were used demonstrates that herd-specific environmental effects can influence the results of biomarker discovery. How significant this is ultimately depends on the research question being asked. If the study aim is to identify biomarkers of external phenotypes , then the impact of environmental effects is important and must be captured. However, if the aim is to identify biomarkers indicative of inter-animal differences free of environmental confounding, or to understand biological processes, our results suggest that the influence of environmental effects could lead to erroneous results. This is consistent with the findings of Posma et al.  who showWe believe that our approach has particular application in animal breeding, where the aim is to understand the biological processes that underpin economically important traits  and to iAll procedures undertaken in this study were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes . Approval to proceed was granted by the Agricultural Research and Extension Animal Ethics Committee of the Department of Jobs, Precincts and Resources Animal Ethics Committee , and the Tasmanian Department of Primary Industries, Parks, Water and Environment . AEC project approval codes 2017-05 and 2018-07.A single 10 mL blood sample was taken from 708 clinical healthy cows, located on 13 farms in south-eastern Australia between September 2017 and July 2019. All cows had been calved 30 days or less at the time of sampling. Cows on all farms except Farm 1 were Australian Holstein-Friesians, while cows on Farm 1 were crossbred animals . All farms operated a feeding system reliant on grazed pasture plus other forages, and concentrates fed in the bail at milking time. Blood samples were collected from the coccygeal vein into 10 mL serum clot activator vacutainer tubes . Samples were allowed to clot at room temperature, before being centrifuged at 1000 g for 20 min at 20 °C. Sera were divided into two aliquots. The first aliquot was refrigerated at 4 °C then shipped on ice to a commercial laboratory for BHBA analysis. The second aliquot was stored at –20 °C until processing for NMR spectroscopy.Serum BHBA concentrations were determined using a colorimetric enzymatic kinetic assay . All ass2O, 98%) were purchased from Cambridge Isotope Laboratories, Inc. .Methanol (>99.9% pure) and dipotassium hydrogen phosphate (anhydrous) were purchased from Fisher Chemical . Sodium 2,2-dimethyl- 2-silapentane-5-sulfonate  and deuterium oxide (D2O phosphate buffer solution (100 mM K2HPO4) containing 0.25 mM DSS-d6 as an internal standard. A 550 µL aliquot was transferred to 5 mm NMR tube for analysis.Serum samples were thawed at room temperature for one hour and were prepared for NMR spectroscopy using a methanol protein precipitation method described by Nagana Gowda and Raftery . BrieflyRoutine 1D proton spectra were obtained on a Bruker Ascend 700 MHz spectrometer equipped with cryoprobe and SampleJet automatic sample changer . A Bruker noesypr1d pulse sequence was used over –0.76 ppm to 10.32 ppm spectral range with 256 scans collected after eight dummy scans at 298K, with a total acquisition time of 2.11 seconds per increment and a relaxation delay (D1) of 2.00 seconds. The overall number of data points was 32,768. A line broadening of 0.3 Hz was applied to all spectra prior to Fourier transformation. Spectra were manually phased then baseline corrected in Topspin v.3.6.1 . Samples were referenced to the internal standard (DSS-d6) at δ 0.00.NMR spectra were imported into MatLab v.R2017b  using the ProMetab v.1.1 script . Each raStatistical analyses were performed in MatLab utilizing the PLS Toolbox v. 8.5.2 . The spectral region containing the residual water peak (δ 4.68–5.00) was removed. Spectra were aligned using the correlation optimized warping algorithm  to accouFor multivariate analyses, unsupervised principal component analysis (PCA) was used. Peaks of interest were identified using the Chenomx NMR suite software v.8.4 , comparison to the literature, and 2D NMR analysis.In order to investigate the effects on spectra of systematic environmental effects  spectra were “corrected” using linear regression models. When correcting for a single categorical fixed effect, this is equivalent to scaling data using the “class centering” pre-processing step. Rather than mean centering, which involves subtracting the global mean from each variable, class centering subtracts the mean of each class. This allows investigation of intra-class variation by removing the effects of inter-class variation . The advThe approach we took was based on the principals of quantitative genetic models, whereIn this study, we only want to remove the effect of environmental factors (as it is the variation in NMR spectra under genetic influence that we are interested in), so the equation can be further simplified toThe “corrected phenotype”  is defined as the residuals from the above model. For the purposes of this study each chemical shift was treated as a separate phenotype, with the signal intensity at each chemical shift being an individual phenotypic observation. The “corrected spectra” was a matrix of the residuals of each model. A 707 × 24,349 matrix of signal intensities of pre-processed spectra was imported into the R statistical software package v 3.6.2 . Each ro2 values from each regression model were stored in a separate vector. This resulted in four vectors each containing 24,349 R2 values; each value representing the percentage of variation in signal intensity explained by the fixed effect(s) at a given chemical shift.The RA separate PCA was performed on each of the 4 corrected spectral datasets (as described in 4.7). Scores of the first three PCs were extracted for each model, and for the PCA model constructed using uncorrected data. We then calculated Pearson’s correlations between scores derived from the 5 PCAs using the corrplot package  in R v 3An alternative approach to investigating the influence of fixed effects is to use multiple linear regression on PC scores from uncorrected spectra. The advantage of this approach is that all fixed effects can be fitted simultaneously, and the statistical significance of each fixed effect can be calculated. The model used wasnted for .Finally, we validated our results using the analysis of variance (ANOVA) simultaneous component analysis (ASCA) method in the PLS Toolbox . ASCA is1H NMR spectra to serum BHBA concentrations determined by colorimetric assay. The aims of this analysis were (1) to assess the robustness of OPLS models built using uncorrected data and (2) investigate the influence of systematic environmental effects on the interpretation of 1H NMR spectra when used for untargeted metabolomic analyses.In order to assess the utility of large and diverse datasets in livestock metabolomics studies, we used orthogonal partial least squares (OPLS) regression to compare 2 and RMSE of calibration, cross validation , and external validation. The statistical significance of OPLS models was determined using permutation testing . Only uncorrected data were used for this part of the analysis.The robustness of OPLS models constructed using large and diverse datasets was assessed using a leave-one-farm-out external validation. This involved setting aside data from one farm, training OPLS models using data from the remaining 12 farms, then using the withheld data for external validation. This process was repeated until data from each farm was used as an external validation set once. Model performance was assessed using the RTo assess the impact of fixed effects on the results of untargeted metabolomic analyses we compared the results of OPLS models constructed from (1) uncorrected data from Farm 1 only (N = 129), (2) uncorrected data from all farms, and (3) data from all farms corrected for all fixed effects (Model 4). Farm 1 data was used to simulate a more “typical” metabolomics experiment in which confounding from environmental effects is controlled through experimental design. When corrected spectra were used, reference BHBA concentrations were corrected for the same fixed effects (Model 4). The residuals of this model represent the “corrected BHBA” concentration, which is the expected BHBA concentration of an individual accounting for differences in WIM, Parity and Herd. This poses some challenges in terms of interpretation, as negative residual values  are possible. However, for the purposes of genetic evaluations, the ranking of an animal, or the relative phenotypic value, is of more interest than an absolute value. The corrected value can therefore be considered a “corrected phenotypic ranking.”1H NMR spectral features. Variable importance of projection (VIP) scores were used to identify the most statistically significant spectral features in each model. Variables with VIP scores greater than one were considered significant [The impact of fixed effects on the ability of NMR spectra to predict external phenotypes  was assessed by comparing the predictive ability of OPLS models. The influences of fixed effects on biomarker discovery were investigated using scores and loadings on LV1 which show the magnitude and direction of relationships between BHBA concentration and nificant .1H NMR serum metabolomic profiling of clinically healthy dairy cows in early lactation. In particular, we investigated the effects of systematic environmental factors on the serum metabolome. We used linear regression to correct spectra for (1) herd of origin; (2) parity; (3) WIM; and (4) herd, parity, and WIM simultaneously. Corrected and uncorrected spectra were then analyzed using PCA. Comparison of PCA results showed that herd of origin had a much greater impact on the serum metabolome than either parity or WIM. In order to simulate the impact of these effects in untargeted metabolomics, we used OPLS regression to quantify the relationship between both corrected and uncorrected NMR spectra, and the current gold-standard biomarker of energy balance in dairy cows, BHBA. Our results showed that (1) models constructed using uncorrected data from multiple farms provided reasonably robust predictions of serum BHBA concentration, (2) environmental effects can alter the results of biomarker discovery, and (3) that correcting spectra for environmental effects using linear regression may be useful when the aim of analysis is to investigate phenotypic variation free of confounding from environmental effects .In this study we investigated the feasibility of using large and diverse datasets for untargeted


Labrus berggylta). Using an in vitro model, we differentiate how signals initiated by physical stretch (cellulose and plastic beads) and nutrients (lipid and protein) modulate the gut evacuation rate, motility patterns and the transcriptome. Intestinal stretch generated by inert cellulose initiated a faster evacuation of digesta out of the anterior intestine compared to digestible protein and lipid. Stretch on the intestine upregulated genes associated with increased muscle activity, whereas nutrients stimulated increased expression of several neuropeptides and receptors which are directly involved in gut motility regulation. Although administration of protein and lipid resulted in similar bulbous evacuation times, differences in intestinal motility, transit between the segments and gene expression between the two were observed. Lipid induced increased frequency of ripples and standing contraction in the middle section of the intestine compared to the protein group. We suggest that this difference in motility was modulated by factors  that are known to inhibit gastrointestinal motility and were upregulated by protein and not lipid. Our findings show that physical pressure in the intestine initiate contractions propelling the bolus distally, directly towards the exit, whereas the stimuli from nutrients modulates the motility to prolong the residence time of digesta in the digestive tract for optimal digestion.The effects of nutrient and mechanical sensing on gut motility and intestinal metabolism in lower vertebrates remains largely unknown. Here we present the transcriptome response to luminal stimulation by nutrients and an inert bolus on nutrient response pathways and also the response on gut motility in a stomachless fish with a short digestive tract; the ballan wrasse ( Ingestion of food initiates a cascade of neural and hormonal signals that prepare the body, and the digestive system in particular, for the tasks ahead and also continues to regulate digestion until the meal has been completely processed . Digestii) non-propulsive contractions, such as segmentation, and ii) propulsive contractions, such as peristalsis and migrating motor complexes (MMCs)  before administration of the bolus into the intestine. Cellulose mix was created using 0.5 mg cellulose  in 1.5 mL H2O, adding a tiny amount of Brillant Blue  as a color maker. These five mixes were made and stored at– 20 °C for use within a week. The premade mixes were thawed and warmed up at 14 °C before being administered as a single bolus into the intestines. The plastic beads were rinsed in ddH2O before being administered.Six treatment mixtures (mix) were prepared, four contained nutrients: intact lipid (IL), hydrolyzed lipid (HL), intact protein (IP), hydrolyzed protein (HP), and two with inert matter: cellulose (CL) and plastic beads (PB). Intact lipid mix was made by stirring a mixture of 80% (by volume) cod liver oil  . Fish wet al. . The empty bulbous was assumed to represent an average width which was equal to or smaller than α and named as “bulbous emptying width—BEW”. The BEW values occurred at various points in time during the 14-hour period. Thus, the bulbous emptying time (BET) was defined as the time when the first BEW value occurred after starting to record the treatments.The bulbous is a morphologically distinct feature of the anterior gut of the ballan wrasse, with an expanded diameter relative to more posterior regions . Here weet al. . Brieflyeos ① in  and calin et al.   to BET, Bulbus emptying time, and period II was from BET to 14h post-starting point. Motility patterns in each period were analyzed from three 30 min. sub-periods based on the experimental design in . The fir-1 , above) for analysis of motility patterns. The classification builds on our previous study where we characterized the motility patterns of ballan wrasse intestines into three types of contractions; standing contractions, ripples, and slow propulsive contractions . The mixes of samples and buffer sample were heated 5 min, 95 °C. The samples and MW-marker (BioRad#161–0375) were loaded into wells of precast 10% SDS-gels (BioRad #456–1043) in Tris-glycine SDS Running Buffer  using a BioRad MiniProtean®Cell according to the manufacturers instruction. The electrophoresis was run for 30 min. at a voltage of 200 V. The sorted proteins in the SDS-gels were incubated with fixing solution  overnight with gentle agitation at room temperature before changing to a staining solution . The staining process was run for 20 min. and followed by destaining in a solution of 40% methanol and 10% glacial acetic acid changing four times for 5, 30, 60, and 10 min. each. Amersham ECL™Western Blot Analysis System  and Chemi Chemiluminiscence Image Capture  were used to detect proteins. Signal strength of each specific band was calculated using Gene Tools from Syngene, file version 4.03.10, Synoptics Ltd. The intact protein bolus was made of casein  which consists of proteins weighting from 19000–23700 Da. The image shows that protein compositions in the feces between intact and hydrolyzed protein were similar and they were similar to the protein compositions of the hydrolyzed protein bolus. The presence of proteins weighting 6500–14400 Da in the feces in the intact protein treatment suggests that the digestion of protein in the intestines was not successful blocked by the protease inhibitor.Lane 1, protein standard with multiple molecular weights ; lanes 2, 4, 6, 8, and 10, feces collected from five individual intestines at 14 h after fed a bolus of intact protein; lanes 3, 5, 7, and 9, feces collected from four individual intestines at 14 h after fed a bolus of hydrolyzed protein; lanes 11 and 12, hydrolyzed protein bolus. The samples (nutrient bolus and feces) were diluted 16 times in dH(TIF)Click here for additional data file.S2 FigFFA, free fatty acids; TAG, triacylglycerol. The compositions of lipid were determined using chromatography with the 19:0 methyl ester as an internal standard according to Lie and Lambertsen 1991 . There w(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.