Oil mixture rich in polyunsaturated fatty acids supplemented to low- or high-starch diets reduced dry matter intake and increased energy digestibility of lactating cows. High starch level improved nutrient digestibility and tended to reduce ruminal acetate:propionate ratio but did not affect rumen pH, molar propionate ratio, or ruminal CH4 emissions. Oil decreased absolute ruminal CH4 emission or tended to decrease CH4 per energy corrected milk.Methane produced by ruminants contributes to increased greenhouse gas effect. There are various nutritional strategies to reduce methane emission, such as supplementing fat or changing starch levels in the diet. Understanding the interactions of these strategies on methane emission, as well as performance, digestibility, and rumen fermentation is important. The present study aimed to assess the effects of starch level with or without a mixture of sunflower and fish oils on nutrient intake and digestibility, milk yield and composition, rumen fermentation, ruminal CH4) emissions, and microbial diversity. Experimental treatments comprised high (HS) or low (LS) levels of starch containing 0 or 30 g of a mixture of sunflower and fish oils (2:1 w/w) per kg diet DM . Intake of DM did not differ between cows fed LS and HS diets while oil supplementation reduced DM intake. Dietary treatments did not affect milk and energy corrected milk yields. There was a tendency to have a lower milk fat concentration due to HSO compared with other treatments. Both high starch level and oil supplementation increased digestibility of gross energy. Cows receiving HS diets had higher levels of total rumen VFA while acetate was lower than LS without any differences in rumen pH, or ruminal CH4 emissions. Although dietary oil supplementation had no impact on rumen fermentation, decreased CH4 emissions (g/day and g/kg milk) were observed with a concomitant increase in Anoplodinium-Diplodinium sp. and Epidinium sp. but a decrease in Christensenellaceae, Ruminococcus sp., Methanobrevibacter ruminantium and Mbb. gottschalkii clades.Four multiparous dairy cows were used in a 4 × 4 Latin square to examine how starch level and oil mixture impact dry matter (DM) intake and digestibility, milk yield and composition, rumen fermentation, ruminal methane (CH Additionally, it has been shown that 5–7% of gross energy (GE) intake is lost through CH4 production from dairy cows [4 mitigation [4 reducing strategy [The growing human population is boosting the demand for milk and meat as sources of animal protein, resulting in several challenges for ruminant production systems, including the need to reduce their contribution to greenhouse gas emissions. This calls for special attention to solutions for reducing methane  also affect the number of methanogens while unsaturated fatty acids (linoleic and α-linolenic acids) shift rumen fermentation towards production of propionate and, therefore, reduce CH4 production through toxic effects on cellulolytic bacteria and protozoa [On the other hand, unsaturated fat supplementation is another feeding strategy which not only reduces enteric CHoduction ,2 but caoduction  with potoduction . Unlike 4 production in dairy cows. Therefore, the present study aimed to assess the effects of starch level with or without a mixture of unsaturated fatty acids (sunflower and fish oils) on nutrient intake and digestibility, milk yield and composition, rumen fermentation, ruminal CH4 emissions, and microbial ecology in dairy cows.Thus, our hypothesis was that higher starch level and oil supplementation would have additive effects on reducing ruminal CHAll experimental procedures were approved by the National Ethics Committee  in accordance with the guidelines established by the European Community Council Directive 86/609/EEC [w/w)/kg diet DM   producing 35.2 ± 2.10 kg milk/d. The cows were fitted with rumen cannula  and each experimental period consisted of 14 days diet adaptation, five days as sampling period, and a 16-day washout to avoid carry-over effects to the next period. The cows were randomly allocated to the diets. Diets, formulated to be isonitrogenous, were used based on grass silage (forage to concentrate ratio 55:45 on a dry matter (DM) basis) consisting of low starch (LS) or high starch (HS) levels (16.1 and 202 g/kg DM) with 0 or 30 g of unsaturated fatty acid mixture  . Sugar bThe oils were stored in +4 °C until incorporated into the low-or high-starch TMR to avoid oxidation of unsaturated fatty acids and the oil replaced concentrate ingredients. The oil mixture and starch levels were selected based on our previous experiences  where saDaily feed intake was measured by subtracting the refusals from the offered feed throughout the study but intakes during d 14–17 of each experimental period were used for statistical analysis. Representative samples of silage and concentrate ingredients during the sample collection period were used for chemical analysis. The samples were pooled within each period before chemical analysis using the standard methods described by Shingfield et al. . In addin = 8) through the rumen cannula. Then, pH was measured using a portable pH meter . Rumen liquid was filtered through two layers of cheesecloth and 5.0 mL ruminal fluid was preserved with 0.5 mL of saturated HgCl2 and 2.0 mL of 1 M NaOH to determine VFA. Furthermore, ammonia-N concentration was analyzed by collecting additional ruminal fluid (15.0 mL) preserved with 0.3 mL of 50% (vol/vol) sulfuric acid. The ruminal samples were stored at −20 °C until the time of analysis. Analysis of VFA and ammonia-N were performed as described by Shingfield et al. [On d 18 of each period and at 1.5 h intervals from 06:00 until 16:30 h, a suction pump with a Büchner flask was used to collect samples of ruminal fluid . The chemical composition of fecal samples was determined using the same methods for the feed samples as described earlier.4 and carbon dioxide (CO2) emissions were recorded over 6 days period (d 11–17 of each period) using sulfur hexafluoride (SF6) as a tracer marker as described by Bayat et al. [6, CH4 and CO2 concentrations because cows were housed in a well-ventilated facility (72 m3/min) and fitted with custom-made sponges placed between the outer edge of the cannula flange and the abdominal wall to minimize the exchange of surrounding air with ruminal contents. Gas chromatography  proposed by Regina and Alakukku [4, CO2, and SF6 concentrations. Actual release rate of SF6 (1.16 ± 0.19 mg/d) in the rumen during the experiment as well as the concentrations of CH4, CO2, and SF6 measured by GC were used to calculate daily ruminal CH4 and CO2 emissions as following:Ruminal CHt et al.  and valit et al. . BrieflyAlakukku  was applSamples of ruminal digesta (2 kg) were collected from four sites  during each period on d 15 at 15:00 h and d 17 at 09:00 h. Samples were mixed and 50 g subsample was placed into a plastic bag and snap-frozen in liquid nitrogen. Samples were stored at −80 °C until DNA extraction. Total DNA was extracted from 250 mg of ruminal digesta following Yu and Morrison . Sequenc4 were calculated using the factor 55.24 kJ/g [2) emissions as proportions of organic matter intake (OMI), organic matter digestibility (OMD), and milk and ECM yields, were calculated by dividing daily CH4 emissions (g/d) by OMI, OMD, and milk and ECM yields, respectively. Methane emissions as percentage of GE intake (GEI) was calculated as CH4 energy (MJ/d) by GEI (MJ/d).The difference between nutrient intake and fecal outputs was used to calculate total tract digestibility coefficients. Energy losses as CH.24 kJ/g . Potenti.24 kJ/g . Methanep ≤ 0.05 was used to determine significant effects of starch, oil, and their interaction. In addition, probabilities at 0.05 < p < 0.10 were considered as a trend.Experimental data was analyzed by ANOVA for a 4 × 4 Latin square with a 2 × 2 factorial arrangement of treatments through the mixed procedure of SAS  with a model that included fixed effects of period, starch level, oil level, and starch by oil interaction, and the random effect of cows assuming an autoregressive covariance structure fitted on the basis of Akaike information and Schwarz Bayesian model-fit criteria. The averages of data for cow within period were calculated before statistical analysis. The values reported are least square means ± SEM. The significance level 2 transformation to account for the non-normal distribution of taxonomic count data, as implemented in Calypso [p < 0.05. p values were further adjusted for the false discovery rate (FDR).For microbial community analysis, taxa with less than 0.01% relative abundance across all samples were filtered out before further analyses. Data was normalized by cumulative-sum scaling and log Calypso . Microbip ≥ 0.18) between cows fed LS and HS diets while oil supplementation reduced (p < 0.01) DM and GE intakes (p = 0.087) OM intake while oil supplementation reduced (p < 0.01) OM intake. Starch intake was much greater for HS compared with LS diets as planned by design, but dietary oil inclusion reduced starch intake more in HS diet (p < 0.01 for the interaction of starch and oil). Intakes of NDF and water-soluble carbohydrates (WSC) were greater (p < 0.01) in cows fed LS than those fed HS diets. Intake of CP in cows fed HS diet was slightly greater (p < 0.05) and again oil supplementation led to a lower (p < 0.01) CP intake. As expected, intakes of saturated fatty acids (SFA), MUFA, and PUFA were greater (p < 0.01) with oil supplementation of both low- and high-starch diets. Milk and ECM yields were not influenced (p ≥ 0.11) by dietary treatments (p = 0.07 for interaction of starch level and oil supplementation) to cause lower milk fat concentration due to HSO compared with other treatments and oil supplementation reduced (p < 0.01) milk protein concentration. Inclusion of oil in the diet tended to reduce (p = 0.087) milk fat yield and reduced (p < 0.05) milk protein yield. Milk production efficiency expressed as ECM/DMI was not affected (p ≥ 0.28) by dietary treatments.Intakes of DM and GE did not differ ( intakes . In compeatments . Howeverp < 0.01) apparent total tract digestibility of DM, OM and starch while decreased (p < 0.01) NDF and pdNDF digestibility (p ≤ 0.02) digestibility of GE and tended to increase (p = 0.058) CP digestibility. There was no interaction between starch level and oil supplementation for any of digestibility measurements.The high starch level, but not oil supplementation, increased (tibility . Both hip > 0.05) on rumen pH while HS diet tended to increase (p = 0.056) total VFA concentration compared with LS diets (p = 0.21) was observed in total VFA as a result of oil supplementation. Compared with the cows receiving LS diet, cows fed HS diets had lower (p < 0.01) molar proportion of acetate and greater (p < 0.05) molar proportions of butyrate, isobutyrate, valerate, isovalerate, and caproate. The experimental treatments did not influence (p ≥ 0.18) molar proportion for propionate. Acetate to propionate ratio tended to be lower (p = 0.07) for HS compared with LS diets. Ruminal ammonia-N was greater for HS compared with LS diets (p < 0.01), and dietary oil inclusion increased ammonia more in HS diet (p < 0.05 for the interaction of starch and oil).The experimental treatments had no impact (LS diets . Howeverp = 0.05) daily ruminal CH4 emissions  and g/kg ECM (p = 0.067) than their control counterparts. No difference (p ≥ 0.15) was found between the treatments in terms of CH4 emissions calculated as proportion of GE intake or g/kg OM digested. The experimental treatments were not different (p ≥ 0.16) in terms of daily ruminal CO2 emission and g/kg OM digested.Inclusion of the oil mixture in LS and HS diets reduced .Ruminal COPrevotella was predominant in all dietary groups (40–50%) with other more abundant genera being unclassified Succinivibrionaceae (0–11%), unclassified Clostridiales (5–6%), Treponema (1–5%), unclassified Ruminococcaceae (3%), unclassified Lachnospiraceae (2–4%), Succiniclasticum (2%), Ruminococcus, Fibrobacter, CF231 and Butyrivibrio . The remaining genera were detected at an abundance below 1%.Sequencing yielded 5222–7362 good quality sequences per sample for bacteria, 1730–18,249 for archaea and 3339–12,123 for ciliate protozoa. Rumen bacterial community was represented by 18 phyla. Bacteroidetes (51–60%), Firmicutes (20–25%), Proteobacteria (1–12%), and Spirochaetes (0.9–5%) were the dominating phyla. Remaining phyla were detected at the abundance below 1%. Among bacterial genera, Methanobrevibacter gottschalkii (50–63%) and Methanobrevibacter ruminantium (12–24%) in all the groups. Other more dominant archaea were Methanosphaera sp. ISO3F5 (6–24%), Methanimicrococcus blatticola (1–15%) and Methanobacterium alkaliphilum (1–3%). Archaea groups belonging to the Methanomassiliicoccaceae (Mmc) family were observed at the abundance below 1%.The Archaea community was dominated by Entodinium (35–56%) in all dietary groups. Other predominant ciliate genera detected at the abundance above 5% in at least one of the diets were: Polyplastron, Ostracodinium, Metadinium, Isotricha, Eudiplodinium-Eremoplastron, Epidinium, Charonina, and Anoplodinium-Diplodinium.Ciliate protozoa community was dominated by p = 0.01) reduced in high starch diets and archaeal richness tended to be numerically lower (p = 0.06) in dietary treatments with oil additive. No significant diet, oil or starch effect was observed on richness of ciliate protozoa  and starch (p = 0.006). Amount of starch in the diet was also a significant explanatory factor (p = 0.05) for the clustering of ciliate protozoa. No distinct clustering of archaea was identified due to diet, oil or starch. Beta dispersion was significantly different between high and low starch bacterial communities (p = 0.009).Beta diversity analysis was performed using RDA and ANOSIM, and identified significant clustering of bacteria with respect to diet (p < 0.05) increased abundance of Anaerovibrio sp., (Spirochaetes) PL-11B10 and ciliate protozoa Eudiplodinium-Eremoplastron (AB536716). Oil caused a significant reduction in (Bacteroidetes) RF16, (Proteobacteria) GMD14H09, Anaeroplasma sp., Prevotellaceae, (TM7) F16, Bacteroidales, and archaea belonging to Mmc. Group 8 sp. WGK1. However, only increase in Anaerovibrio sp. remained significant after correction for multiple testing using false discovery rate (FRD = 0.035) (Diets with oil additive significantly (= 0.035) .Succinivibrionaceae spp., Ruminobacter sp., Selenomonas sp., Moryella sp., Ruminococcus flavefaciens, and ciliate protozoa Isotricha sp. and Entodinium sp. Contrary, diets with low starch content were significantly enriched in Treponema sp., (Bacteroidetes) F16, SR1, Lachnospira sp., Clostridium sp., Acholeplasmatales, Desulfovibrio sp., (Tenericutes) RF39, (Actinobacteria) OPB41, Lactobacillus sp. and ciliates affiliated with Eudiplodinium-Eremoplastron (OTU12), Charonina ventriculi, Ostracodinium sp. and Dasytricha sp. After multiple testing correction, only (Bacteroidetes) S24-7, Succinivibrionaceae sp., Isotricha sp. and Eudiplodinium-Eremoplastron (OTU12) retained significant differences (Diets with high starch content had significantly higher relative abundance of bacteria (Bacteroidetes) S24-7, ferences .p < 0.05) effect on 27 bacterial, one archaeal and seven ciliate protozoan taxa, but after FDR correction, only seven bacterial and two ciliate protozoan taxa remained significant  was significantly more abundant in LS compared with HS or HSO diets, while Isotricha sp. was significantly more abundant in HS compared with LS or LSO diets. Bacteria (Bacteroidetes) F16, (Proteobacteria) GMD14H09, (Bacteroidetes) RF16 and SR1 were detected at the lowest abundance in HSO diet and showed significant differences between HSO and all other diets. Bacteria (Bacteroidetes) S24-7 had highest abundance in HSO diet and showed significant differences in pairwise comparisons with LS and LSO diets. Among archaea Mmc. Group 8 sp. WGK1 was more abundant  in HS diet compared with HSO or LSO diets (Diet composition had significant (nificant . In pairSO diets .p < 0.05), eight bacterial and 10 ciliate protozoan correlations remained significant after correction for multiple testing using false discovery rate (FDR < 0.1).To look at the associations between rumen microbiome and rumen fermentation as well as methane production traits, we applied Spearman correlations on the microbial taxa present in all samples (n = 16 observations). From 50 bacterial, two archaeal and 25 ciliate protozoan significant associations (Clostridium sp. (FDR = 0.035), ciliates Dasytricha sp. (FDR = 0.053), and Charonina ventriculi (FDR = 0.057) (Moryella sp. (FDR = 0.049) but negatively associated with Treponema sp. (FDR = 0.044), (Bacteroidetes) RF16 (FDR = 0.049), Clostridium sp. (FDR = 0.049), and Charonina ventriculi (FDR = 0.07). Charonina ventriculi was negatively correlated with butyrate (FDR = 0.043) while Epidinium sp. was negatively correlated with isobutyrate (FDR = 0.062) and isovalerate (FDR = 0.016). On the contrary, Entodinium sp and Entodinium caudatum were positively correlated with isovalerate . Anoplodinium-Diplodinium sp. and Epidinium sp. were negatively correlated with daily CH4 emissions (FDR = 0.064), while Christensenellaceae and Ruminococcus sp. correlation was positive. Ruminococcus flavefaciens was positively correlated with valerate (FDR = 0.063). Methanobrevibacter ruminantium and Mbb. gottschalkii clades were both positively correlated with CH4 intensity (g/kg milk)  (An increase in acetate was negatively correlated with (Bacteroidetes) S24-7 (FDR = 0.005), but positively with = 0.057) . Increas = 0.12) .The results of this experiment showed that diets with oils rich in PUFA in a moderate amount reduced DM intake, which in turn resulted in lower intakes of other nutrients . Similar observations have been reported previously in experiments with PUFA-rich plant oils ,29, fishThe lack of responses on milk yield due to dietary starch level and unsaturated oil mixture were expected as the diets were designed based on our previous experiences  to causeThe lack of oil supplementation effect on nutrient digestibility, with exception of CP and GE digestibility is consistent with some previous findings ,37,38.While in our experiment high-starch diet did not affect DM intake, an increase was observed for CP, OM, and starch intakes with drops in NDF and WSC intakes, which are due to differences in nutrient contents of the diets. The results of our study are consistent with the findings of Pirondini et al.  and PhilRuminobacter sp. or Selenomonas sp. increased in high starch diets while the abundance of amylolytic Prevotella or cellulolytic bacteria Ruminococcus and Fibrobacter in LS-fed cows was not significantly different from that in HS-fed cows. Another explanation might be that observed differences in fiber digestibility reflect changes in the composition of diets; in HS diet rolled barley and ground wheat were used instead of sugar beet pulp and barley feed which might have different fiber characteristics.In our experiment, digestibility of DM, OM, starch, and GE was higher and CP digestibility tended to be higher in cows fed HS than those receiving LS diets. Beckman and Weiss  observedIt should be noted, however, that in some experiments where starchy feeds were replaced by fibrous by-products, there has been a confounding effect of both starch:NDF ratio and forage proportion of the diet . However4 production compared with non-supplemented diets . This is in line with the findings of the experiments that used oilseeds [4 emission (g/d) reduced by on average 4.7% for each 10 g/kg DM unsaturated oil in the diet. Similarly, Martin et al. [4 production reduces by 3.8% as a result of every 10 g/kg increase in dietary lipid supplementation. The most of this response in our experiment was caused by lower feed intake as CH4 yield (g/kg OMI) was not affected by the oil supplementation . Ramin and Huhtanen [4 yield by 0.043 L/kg of DM. The equivalent value in our experiment was on average 0.045 g/kg of OM intake which is very close to the reported value. Apparently both biohydrogenation of unsaturated fatty acids and amount of unfermentable organic matter introduced by oil to the diet should have caused the lower CH4 yield. Oil potential in reducing CH4 production seems to be a function of factors such as source of oil, fatty acid composition and level of supplementation in the diet [4 production including reduced OM fermentation in the rumen, unfavorable effects of C12:0 and C14:0 on protozoa community, and inhibition of methanogens by 18-carbon unsaturated fatty acids [4 production is concerned, MUFA and PUFA are more effective than SFA in the diet. In this experiment, oil supplementation to both low- and high-starch diets resulted in higher intakes of both MUFA and PUFA compared with non-supplemented diets.The oil supplementation to both low- and high-starch diets resulted in lower CHoilseeds ,46,47, ooilseeds  to supplHuhtanen  in a metty acids ,50 and tty acids  reported4 yield for cows fed oil-supplemented diets in our experiment is consistent with the lack of differences in rumen pH, acetate, propionate, and VFA concentration and acetate:propionate ratio, although acetate and propionate concentrations and acetate:propionate ratio decreased numerically. However, oil supplementation is expected to not only reduce CH4 production but also lower acetate:propionate ratio. The non-significant change in acetate:propionate ratio observed in our experiment can be attributed to the lower DM and concentrate intakes, and the forage portion of our experimental grass silage-based diet. Changes in rumen fermentation pattern due to oil supplementation with diets based on restrictively fermented grass silage (using the silage additive based on formic acid) may be resistant to lipid supplementation [Unaffected CHentation .4 production as a percentage of GE intake while CH4 intensity calculated as g/kg milk or ECM decreased and tended to decrease by oil supplementation, respectively. In fact, the reduction in CH4 intensity indicates that net energy is partitioned more towards milk production, leading to lower CH4 intensity [4 production as a percentage of GE intake for un-supplemented diets  having DM intake of 23.0 and 23.5 kg/d are higher than 6.4% measured from dairy cows receiving rather similar diets with similar DM intake in respiration chambers .Even though the intake of GE increased as a result of including oil in LS and HS diets, there were no significant differences between experimental treatments in terms of CHntensity . The val4 and CO2 production (g/d) and emission intensity (g/kg milk yield). Previous studies [4 production and CH4 emission intensity with the exception of Hatew et al. [4 emission intensity due to increasing dietary starch level. Increased starch content influenced rumen fermentation parameters, with a significant decrease in molar proportion of acetate and a tendency to reduce acetate:propionate ratio. Although high starch diets are expected to reduce acetate and increase propionate molar proportions and to lower CH4 production [Our findings showed that starch level did not influence ruminal CH studies ,40,41 ha4 production, with each type of oil having a different impact on bacterial community. Furthermore, Martin et al. [4 production without a notable change in abundance of rumen methanogens and cellulolytic bacteria. Looking at individual microbial taxa, addition of oil to both low- and high starch diets in this study, provided a suitable ecological niche for lipid hydrolyzing Anaerovibrio sp. [Eudiplodinium-Eremoplastron (AB536716). Similarly, a significant increase in Eremoplastron dilobum abundance was detected in an in vitro experiment with linseed oil but not rapeseed oil additive [Starch level had stronger effect on bacterial richness compared with those caused by inclusion of oil in the diet. Despite reports of toxic effects of oils on microbial community and, therefore, greater expected changes in the rumen microbial composition , this stn et al.  reportedbrio sp.  and Spirbrio sp.  and outsbrio sp. . Neverthadditive , suggestRuminobacter sp., Selenomonas sp. or ciliate protozoa Isotricha sp. and Entodinium sp. that are involved in the utilization of non-structural polysaccharides and soluble sugars [4 production, suggesting lower H2 availability in these ruminal ecosystems. S24-7 was significantly more abundant in HSO diet, was strongly negatively correlated with acetate and tended (p = 0.054) to be positively (R = 0.49) correlated with propionate concentration in the rumen (data not shown). In addition to S24-7, also Prevotella sp., Moryella sp. and members from Paraprevotellaceae family were positively correlated with propionate concentration in the rumen  F16, (Tenericutes) RF39, and Desulfovibrio were found to be tightly attached to switchgrass [Fibrobacter succinogenes, Treponema bryantii has been shown to utilize soluble sugars released from cellulose degradation [Ostracodinium sp. and Dasytricha sp., are known to contain cellulolytic and hemicellulolytic activities, respectively, and were significantly enriched in LS diets. Charonina ventriculi is a holotrich protozoa not frequently observed in the rumen and with limited information about its metabolism. In our experiment, Charonina ventriculi was significantly enriched in LS diets and was negatively correlated with ammonia-N and butyrate, but positively correlated with acetate concentration in the rumen. Correlation profile of Charonina ventriculi was similar to Treponema sp. and Clostridium sp.  in oil supplemented diets. With higher abundances of bacteria Moryella sp., Anaerovibrio sp., (Bacteroidetes) S24-7, (Spirochaetes) PL11B10, Selenomonas sp., Ruminobacter sp., (Paraprevotellaceae) YRC22 and ciliate protozoa Anoplodinium-Diplodinium which had a tendency to be positively correlated with propionate concentration in the rumen, we can hypothesize having an ecosystem with less hydrogen available for methanogenesis.4 emissions (g/d) were positively associated with reduction in Entodinium caudatum. Although Entodinium was the most abundant ciliate protozoa in all diets and smaller Entodinium spp. have been suggested to contribute more to CH4 production compared with larger protozoa in in vitro studies [Entodinium into OTUs suggests functional versatility and differences in host dependency inside this genus Contrary to our results, Belanche et al. [4 production compared to the natural flora and concluded that holotrichs were responsible for increased methanogenesis more than the entodiniomorphids. In our study, OTU affiliated with Isotricha prostoma was negatively correlated with daily CH4 emissions  reduced DM intake and some ruminal methane emission indices without influencing rumen fermentation characteristics or nutrient digestibility. The findings of this experiment show that feeding more starch originating from concentrate portion instead of fiber at a moderate level in dairy cow diets does not favor lower methane production, and oil supplementation is similarly effective on reducing methane in low- and high-starch diets. Therefore, our hypothesis that starch level and oil supplementation would have synergistic effects on CH4 emission could not be proved as increasing dietary starch level did not influence CH4 emission whereas oil supplementation did. Inclusion of moderate amount of the unsaturated oil mixture in the diet did not have profound toxic effects on bacteria, archaea, or ciliate protozoa, which is in line with the minor effect on methane yield.Overall, the results of this experiment show that starch level modified rumen fermentation and nutrient digestibility without influencing DM intake or methane emissions. Inclusion of unsaturated oil mixture (sunflower and fish oils, 2:1


Communicating research findings is an essential step in the research process. Often, peer-reviewed journals are the forum for such communication, yet many researchers are never taught how to write a publishable scientific paper. In this article, we explain the basic structure of a scientific paper and describe the information that should be included in each section. We also identify common pitfalls for each section and recommend strategies to avoid them. Further, we give advice about target journal selection and authorship. In the The online version of this article (10.1007/s13187-020-01751-z) contains supplementary material, which is available to authorized users. Writing a scientific paper is an important component of the research process, yet researchers often receive little formal training in scientific writing. This is especially true in low-resource settings. In this article, we explain why choosing a target journal is important, give advice about authorship, provide a basic structure for writing each section of a scientific paper, and describe common pitfalls and recommendations for each section. In the We recommend that you select a “target journal” early in the writing process; a “target journal” is the journal to which you plan to submit your paper. Each journal has a set of core readers and you should tailor your writing to this readership. For example, if you plan to submit a manuscript about vaping during pregnancy to a pregnancy-focused journal, you will need to explain what vaping is because readers of this journal may not have a background in this topic. However, if you were to submit that same article to a tobacco journal, you would not need to provide as much background information about vaping.https://www.springer.com/journal/13187/aims-and-scope.Information about a journal’s core readership can be found on its website, usually in a section called “About this journal” or something similar. For example, the Journal of Cancer Education presents such information on the “Aims and Scope” page of its website, which can be found here: Peer reviewer guidelines from your target journal are an additional resource that can help you tailor your writing to the journal and provide additional advice about crafting an effective article . These aEarly in the writing process, identify authors, determine the order of authors, and discuss the responsibilities of each author. Standard author responsibilities have been identified by The International Committee of Medical Journal Editors (ICMJE) . To set In the case where multiple papers will result from a single study, authors should discuss who will author each paper. Additionally, authors should agree on a deadline for each paper and the lead author should take responsibility for producing an initial draft by this deadline.The introduction section should be approximately three to five paragraphs in length. Look at examples from your target journal to decide the appropriate length. This section should include the elements shown in Fig. The purpose of the methods section is twofold: to explain how the study was done in enough detail to enable its replication and to provide enough contextual detail to enable readers to understand and interpret the results. In general, the essential elements of a methods section are the following: a description of the setting and participants, the study design and timing, the recruitment and sampling, the data collection process, the dataset, the dependent and independent variables, the covariates, the analytic approach for each research objective, and the ethical approval. The hallmark of an exemplary methods section is the justification of why each method was used. Table The focus of the results section should be associations, or lack thereof, rather than statistical tests. Two considerations should guide your writing here. First, the results should present answers to each part of the research aim. Second, return to the methods section to ensure that the analysis and variables for each result have been explained.Begin the results section by describing the number of participants in the final sample and details such as the number who were approached to participate, the proportion who were eligible and who enrolled, and the number of participants who dropped out. The next part of the results should describe the participant characteristics. After that, you may organize your results by the aim or by putting the most exciting results first. Do not forget to report your non-significant associations. These are still findings.Tables and figures capture the reader’s attention and efficiently communicate your main findings . Each taOpposite the introduction section, the discussion should take the form of a right-side-up triangle beginning with interpretation of your results and moving to general implications Fig. . This seNext, interpret the meaning or explain the significance of your results, lifting the reader’s gaze from the study’s specific findings to more general applications. Then, compare these study findings with other research. Are these findings in agreement or disagreement with those from other studies? Does this study impart additional nuance to well-accepted theories? Situate your findings within the broader context of scientific literature, then explain the pathways or mechanisms that might give rise to, or explain, the results.Journals vary in their approach to strengths and limitations sections: some are embedded paragraphs within the discussion section, while some mandate separate section headings. Keep in mind that every study has strengths and limitations. Candidly reporting yours helps readers to correctly interpret your research findings.The next element of the discussion is a summary of the potential impacts and applications of the research. Should these results be used to optimally design an intervention? Does the work have implications for clinical protocols or public policy? These considerations will help the reader to further grasp the possible impacts of the presented work.Finally, the discussion should conclude with specific suggestions for future work. Here, you have an opportunity to illuminate specific gaps in the literature that compel further study. Avoid the phrase “future research is necessary” because the recommendation is too general to be helpful to readers. Instead, provide substantive and specific recommendations for future studies. Table After you select a target journal, identify the journal’s author guidelines to guide the formatting of your manuscript and references. Author guidelines will often  include instructions for titles, cover letters, and other components of a manuscript submission. Read the guidelines carefully. If you do not follow the guidelines, your article will be sent back to you.Finally, do not submit your paper to more than one journal at a time. Even if this is not explicitly stated in the author guidelines of your target journal, it is considered inappropriate and unprofessional.Your title should invite readers to continue reading beyond the first page , 5. It sBe sure that the title is useful for potential readers searching for your topic. The keywords you select should complement those in your title to maximize the likelihood that a researcher will find your paper through a database search. Avoid using abbreviations in your title unless they are very well known, such as SNP, because it is more likely that someone will use a complete word rather than an abbreviation as a search term to help readers find your paper.After you have written a complete draft, use the checklist Fig.  below toESM 1(PDF 362 kb)


Due to the COVID-19 outbreak, a series of chain reactions, like international trade breakdown, stock market collapse, and crude oil's collapse, have adversely affected the global economy, particularly small and medium-sized enterprises (SMEs). As a result, the Chinese government issued many fiscal and financial policies to support SMEs. This paper analyzes SMEs' coping methods and conceptual changes affected by the epidemic and distinguishes “victims” and “beneficiaries.” Subsequently, based on extensive international experience and local government experience, it provides effective suggestions for the Chinese government to deal with the post-epidemic era's economic changes, policy suggestions, and coping strategies for SMEs' short-term and long-term sustainable development. The global spread of novel coronavirus has caused a huge economic shock that may exceed the global financial crisis of 2008 , which has significantly reduced the financing cost of enterprises. Under the government's guidance, commercial banks have offered more favorable loan rates to SMEs. For example, CCB's interest rate on loans to SMEs in Hubei dropped by 0.5% The Bank of Communications lowered the lending rate by 0.5% for enterprises related to epidemic prevention and further reduced the lending rate for some key enterprises.The Agricultural Bank of China has also offered preferential loan rates to key industries and enterprises related to epidemic prevention. Financial institutions should not blindly withdraw loans, cut off loans or squeeze loans. Due to the epidemic, small and micro enterprises facing repayment difficulties should extend the term, renew loans, reduce unpaid interest and other assistance, and properly arrange repayment plans until the “first-level response” is removed.In general, with the joint efforts of many parties, the financing of small and micro enterprises in 2020 has achieved a significant effect of “quantity increase, price decrease, and scope expansion.” By the end of 2020, the balance of small and microloans of Pratt and Whitney was 15.1 trillion yuan, up 30.3% year on year. In December 2020, the interest rate of newly issued Pratt and Whitney small and microloans was 5.08%, down 0.8 percentage points from the same period in 2019. In 2020, a total of 32.28 million small and micro businesses were supported, up 19.4% year on year.On March 1, 2020, silver was issued by the Ministry of Finance on a temporary extension to micro, small and medium enterprises loan servicing notice, put forward by the principle of marketization, government by law, to comply with conditions, liquidity temporary difficulties of small and medium-sized enterprise loan, giving a brief delay servicing arrangements, the longest can be extended to June 30, 2020.In Hubei province, the worst-hit area, the circular also requires banking financial institutions to provide Hubei with a special credit scale, implement preferential internal fund transfer pricing, and strive to reduce the total financing cost of inclusive small and micro businesses by more than one percentage point from the average level of the previous year in 2020.According to Government of China ,b, the SMany local governments have explicitly proposed government procurement for SMEs to encourage production. For example, in February 2020, the Chengdu municipal government issued 20 policies and measures to effectively deal with the epidemic and stabilize economic operation, which mentioned that the government should take measures such as reservation share, preferential evaluation, and eliminating threshold, and strive to make the proportion of goods and services purchased by SMEs reach more than 80%. In addition, establish and improve the advance payment system, and encourage purchasing units to advance a certain percentage of the contract price to suppliers winning the bid.The preceding discussion indicates that certain SMEs are vulnerable to the epidemic, reducing their future viability and growth potential. In the following part, the relevant support policies issued by the Chinese government and the current situation in China will be analyzed to provide suggestions for the government to introduce more targeted policies and measures to reduce the negative impact of the epidemic crisis on SMEs.Government policies can be divided into long-term and short-term policies from the duration of benefits. In the short term, the government needs to act quickly to ensure that SMEs can continue to operate, ease liquidity constraints for SMEs, and ensure employment stability OECD., . The ChiIn terms of financing, the government proposed to strengthen credit support for manufacturing, small and micro enterprises, private enterprises, and other key areas, strengthen the construction of banking services, maintain the growth of loans, earnestly implement the requirements for reducing the pressure of total financing costs, and increase the supply of medium—and long-term loans to the manufacturing sector.Accordingly, policies have also been introduced to allow small, medium, and micro enterprises temporarily facing liquidity difficulties to delay interest payment and to increase the reloan and rediscount quota of 500 billion yuan for small and medium-sized banks to increase credit support to small, medium, and micro-enterprises, and to reduce the reloan interest rate for agriculture and small by 0.25 percentage points to 2.5% , Wu et al.  simulateKey industries affected by the epidemic can be identified at the national level. In the short term, prudent and accommodative monetary policy can reduce business operating costs and increase the counter-cyclical adjustment of fiscal policy to expand the scale of fiscal spending. At the regional level, the epidemic's impact on the consumption channel is more rapid and direct, which in turn is transmitted to the upstream manufacturing industry. Therefore, while paying attention to the direct impact of the epidemic on SMEs, the government needs to pay attention to the indirect industry of supply chain transmission. At the enterprise level, practical assistance should be provided based on actual production needs, and deregulation should be accompanied by improved regulatory efficiency to stabilize employment and protect people's livelihood.Finally, the implementation of new policies may generate certain economic risks. Economic risk refers to the risk caused by the uncertainty of economic policies on living standards and market supply due to macroeconomic environment changes  to ensure that these large enterprises do not encode government funds for SME employment recovery.Secondly, it should fully play the employment-absorbing capacity of the fast-growing SMEs in the epidemic. Policies should be put in place to connect these fast-growing SMEs and ensure that they can better absorb employment. For example, some economists argue for hiring subsidies to encourage firms to engage more in times of crisis.Thirdly, encourage enterprises to take the initiative to raise financial needs. As mentioned above, the financing difficulties of SMEs not only exist on the supply side but also on the demand side. Many enterprises refuse to actively seek external financing opportunities for fear of financing failure, resulting in a serious shortage of corporate capital. According to the study, capital investment by SMEs will fall significantly under the outbreak Herbane, . At thisFourthly, strengthen financial support for SMEs. In addition to lowering the loan threshold for SMEs by state-owned banks, various social banks and other financing organizations can also be encouraged and rewarded to open the green light for SMEs and launch the capital pool of private enterprises to promote the economic recovery of SMEs. Furthermore, as Brown  suggesteAnother approach is offering free financial advice to small and medium-sized firms from different funding sources, particularly those too small to understand complex financial products fully. Increasing SMEs' access to external financing information and improving their understanding will help them obtain financing suitable for themselves Wilson, .Fifthly, promote the combination of SMEs and online business models. The Chinese government has provided 500 billion yuan of additional loan funds and other preferential financing policies for SMEs. This is currently the practice in most developed countries OECD., . HoweverIn March, China's epidemic prevention and control situation continued to improve, production and living order were steadily restored, and enterprises' resumption of work and presentation significantly accelerated. Although, as a result, China purchasing managers' index fell sharply last month on the base, including manufacturing PMI was 52.0%, up 16.3% the previous month, purchasing managers' index rebounded in March to more than a tipping point is the sharp decline after the rebound in February.Still, more than half of the firms are to return to work, and the production situation has improved since last month. Therefore, it does not represent China's economic operation has returned to normal levels. At the same time, due to the lag of the epidemic and the global spread, the global PMI index gradually fell below 50% from February 2020, reached the lowest 39.8% in April, and slowly recovered from May.In a crisis like this, cash flow from operating activities is reduced because orders are reduced. SMEs are reluctant to take on additional debt because they will not repay their debts on time. Online business models can provide ideas for firms to increase revenue. According to Baidu Index, the popularity of online office searches reached a peak of 1,367 in February.First, promoting social media is conducive to SMEs actively displaying their products or businesses and promoting promotional activities. Second, firms can also build online communities to build customer loyalty and effectively understand customers through new tools such as digital marketing to enhance the customer experience. As a result, policies that focus solely on providing government loans are often flawed. Instead, policies must focus on changing the offline business model of many SMEs to embrace online business and adapt to the epidemic situation.The government should provide necessary online business training to SMEs that need it. For example, according to a report by, the Tianjin Bureau of Commerce has offered online business education for merchants on the WeChat public account “Goutianjin Purchase Tianjin” since February. Such enterprise education and training can be more systematic and refined for different SMEs to provide different content. And information technology offers the necessary support for the online operation of enterprises. The adoption of cloud computing through accounting software is a good example. It reduces the cost of monitoring and accessing the security of financial reports.In addition, enterprises can use information technology to track inventory and create scientific job management to save valuable business time. Moreover, governments must work on digitizing SMEs and making them part of the ecosystem of the networked economy. Many SMEs will also spontaneously transform the development. Therefore, in addition to encouraging SMEs to develop online businesses, the government should consider providing small grants to SMEs interested in taking the initiative to develop online businesses.Providing the right incentives is essential to help SMEs adapt to the new realities. Even a small amount of funding can make a big difference in the behavior of small firms and help them increase their ability to generate revenue. The government can also temporarily increase the pre-tax deduction for capital investment, encourage SMEs to invest in technology, and accelerate their technological and online orientation.The epidemic affects different types of enterprises in other times and degrees of return to work, and March and April 2020 are the time for each industry to return to work and production.Sixth, generate income for SMEs through government purchases. The Chinese government is a huge buyer of goods and services in the domestic market. In 2018, the government procurement scale nationwide reached 3,586.14 billion yuan, accounting for 4% of China's GDP. In addition, central and local governments and other public infrastructure sectors have become important customers for many businesses through spending on vital public services such as schools, hospitals, and prisons.Moreover, the government has ample capacity to adjust its procurement budget. However, it is often large firms that benefit from government procurement. If the government adopts only an open tender model where the lowest bidder wins, small firms will not compete with large ones on cost, leading to large firms signing most of the contracts from the public sector. Therefore, the Chinese government will take care of SMEs in government procurement.In 2018, for example, the total amount of government procurement contracts awarded to small, medium, and micro enterprises reached 2,748.86 billion yuan, accounting for 76.7% of the total amount of government procurement. This proportion can be expanded to support SMEs in the current special circumstances, which are much more difficult than large enterprises. Under the epidemic, the government needs to increase public health services, financial support, and other aspects and increase services and commodities. When conditions permit, the government can hand over these demands to SMEs to produce, providing them with much-needed assistance.Seventh, timely assessment of policy effects and adjustments. Although the epidemic situation in China has gradually stabilized, the international environment is still not optimistic. To cope with this rapidly changing and unpredictable situation, the government should increase official surveys and set up a database. At the same time, reliable social survey data should be used to make timely and accurate assessments of their supporting behaviors, such as providing commercial loans, and timely adjustments should be made based on this. The government should also investigate SMEs to understand their difficulties and concerns and provide targeted help. In addition, the government can also predict future economic development and changes through various research results to track the effectiveness of policies in the present and future , the Natural Science Foundation in Zhejiang Province (Grant number: LQ20G030013) and HDU (Grant number: KYS145619115).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The FCN with the addition of 20% CAS exhibited slow deterioration of textural properties during holding for 30 min. The analysis of the changes in the content of free SH group and glutenin macropolymer (GMP) demonstrated that the addition of CAS promoted protein disulfide cross‐linking and decreased protein mobility during holding. Fourier transform infrared spectroscopy (FT‐IR) revealed that FCN with the addition of CAS had low decrement in α‐helix and β‐sheet during holding, indicating that starch acetates contributed to the maintenance of the gluten network structure.The physicochemical properties of starch acetates with an equal degree of substitution prepared from pea, corn, and wheat starch and their effects on frozen cooked noodle (FCN) quality were investigated. The result showed that the three kinds of starch acetates had different effects on the quality of FCN due to their different blue values, freeze‐thaw stability, and crystalline morphology analyzed by XRD ; acetylated corn starch (CAS), acetylated wheat starch (WAS), and acetylated pea starch (PAS) were effective in reducing cooking loss and improving springiness of FCNs. Starch acetates can be used as a novel additive in FCNs to provide specific functionality and alleviate the deterioration of quality during holding. With the development of society and economy, consumers nowadays put forward higher requirements for ready‐to‐eat foods, such as convenience and health. As an emerging noodle product, frozen cooked noodles (FCNs) are widely accepted by consumers due to a short reheating time before eating. Meanwhile, with the development and application of chilling and quick‐freezing technology, the FCNs will show a great market potential.The two main steps involved in the process of FCNs are precooking and freezing. Precooking involves boiling and steaming, which not only improved the springiness and chewability of boiled noodles but also solved the problems of the dry texture of steamed noodles  were used to prepare starch acetate with an equal degree of substitution (DS) and then applied to the preparation of FCNs. The objective of this study was to investigate different starch acetates in the texture of subsequently cooked noodles to determine whether it was possible to make a new type of convenient and nutritious FCNs.22.1Starches were provided by Henan Hengrui Starch Technology Co. LTD. Wheat flour was purchased from Carrefour supermarket . Pancreatin, amyloglucosidase, and D‐Glucose Assay Kit were procured from Megazyme International Ltd. All chemicals and reagents used here were of analytical grade.2.2The starch sample (30 g) was dispersed in distilled water to make 30% (w/v) starch suspension, which was then adjusted to pH 8.5 with 3% (w/w) NaOH. Specific doses of acetic anhydride were added to the suspension to obtain modified starch with an equal DS. NaOH was used to maintain pH 8.5. Starch samples were placed in a microwave instrument with a cooling water circulating device  and treated with microwaves and ultrasonics simultaneously. The specific operation is as follows: the sample was under microwave and ultrasonic treatment for 5 s, and then just treated by ultrasonics for 55 s. The above process was performed by microwave at 500 w for 100 s and the ultrasonication with a frequency of 24.5 KHz at 500 w for 20 min. The slurry was cooled to room temperature. The obtained starch was allowed to dry in an oven at 45°C for 24 h. The wheat, pea, and corn starches under the coupled treatment by microwave and ultrasonication were named as WAS, PAS, and CAS, respectively.2.32.3.1W is the mass fraction of acetic acid substituents (%), V1 is the volume of HCl required for the titration of the modified starch sample (ml), V2 is the volume of HCl required for the titration of the control sample (ml), C is the molarity of HCl solution (mol/L), and m is the quality of starch (g).The DS of modified starch samples was determined using a titration method  analysis with an X‐ray diffractometer  using Cu‐Kα X‐rays at 1.54056 Å and an angle of diffraction scanning from 5° to 40° with a step length of 0.02°. The relative crystallinity was calculated as the proportion of the crystalline area to the total area multiplied by 100.2.4The flour blends were prepared by mixing wheat flour and starch acetate. The flour blends were poured into a dough mixer container , and an appropriate amount of water containing 1.5% sodium chloride was slowly injected. The mixture was stirred for 2 min at a slow speed and 10 min at a fast speed. The resulting dough was aged at 25°C and 80% relative humidity for 20 min. After resting, the dough was successively compressed several times at different roll gap settings, and the final sheet was cut into noodle strips about 1.0 mm thick and 2.0 mm wide using the noodle machine . Fresh noodles (30 g) were steamed in a steamer for 5 min and then boiled in 500 ml water for 3 min. The cooked noodle was cooled down by immersing it in 500 ml cold water (4°C) for 1 min, drained for 1 min, and put in a plastic bag for freezing in a −40°C refrigerator for 60 min. The frozen noodles were finally stored at −18°C for further analysis. Some fresh noodles and FCNs were freeze‐dried and ground into flour for subsequent analysis.2.5FCNs (20 g) were cooked in boiling water (400 ml) for 1 min. The water absorption of the noodles was determined as the ratio of the added weight of cooked noodles to total initial FCN weight. The collected gruel was transferred into a 500‐ml‐volume bottle, and the volume was fixed at room temperature. One hundred milliliters of the above gruel was precisely transferred into a beaker (predried to constant weight), and then the beaker was dried in an air oven at 105°C to constant weight. The weight of the residue is reported as the percentage of the noodle samples .2.6The textural properties of cooked noodles were measured using a TMS‐PRO texture analyzer . Three cooked noodle strands were arranged adjacent to each other on the operating platform. The test conditions were as follows: texture profile analysis (TPA) mode; probe selection, P/50; parameter settings of pretest speed, test speed, and post‐test speed of TPA were 1.00, 1.00, and 1.00 mm/s, respectively; test compression rate of TPA, 75%; and trigger force, 5 g. Each sample was measured three times.2.7The sulfhydryl group contents of the samples were determined according to the method described by Rakita et al. . The res2.8The determination of GMP was conducted following a method reported by Li, Zhang, et al.  with som2.9−1 to 400 cm−1 with a Thermo Nicolet Fourier transform infrared spectrometer . The samples were subject to 64 scans per sample at a 4 cm−1 resolution. The secondary gluten structure was determined after Gaussian deconvolution and secondary derivation of the amide I region using Peak Fit v4.12 software. Secondary structure content was presented as a percentage of the corresponding area by ratio to the total amide I band area.Fourier transform infrared spectroscopy (FT‐IR) of the sample was conducted, and the results were analyzed according to Jiang et al. . The sam2.10p < .05.Mean values and standard errors of triplicate data were analyzed by ANOVA with SPSS 16.0. The multiple comparisons of data were determined by Ducan's test. Significance was defined at 33.1p < .05) difference in native and treated starches as presented in Table Amylose content is an important characteristic that greatly influences the functionality of starch  in the best reheating time was observed among FCNs with different starch acetates. The addition of three kinds of starch acetates shortened the reheating time of FCNs to varying degrees, and the reheating time gradually decreased with the increase in the amount of starch acetate. It was expected due to the increase in water absorption of noodles by adding the starch acetate, which reduced the energy of starch gelatinization and changed the best reheating time.The cooking parameters, including best reheating time, cooking loss, and water absorption rate of the noodles, are shown in Table The water absorption rate reflects the capacity of water absorbed during cooking. Noodles have an excellent texture only when the water absorption rate is appropriate. The water absorption rate of noodle samples increased with the addition of starch acetate, which might be because the added starch acetates filled the gluten network and enhanced its extensibility. Also, the addition of starch acetate formed certain hydrophilic groups, which improved the hydrophilicity of dough, thus promoting the crosslinking of starch molecules. Furthermore, prolonging the cooking time will lead to an increase in the boiled noodle weight and, therefore, an increase in the water absorption rate.The cooking loss represents the number of particles diffused out from the samples into the noodle soup medium during boiling. A higher cooking loss indicates more dissolution in the noodle soup and worse cooking resistance  above the value of fresh noodles. Although depolymerization of GMP was observed during the frozen storage in a previous study  and amide II (1500–1600 cm−1). Amide I band is further investigated because the absorption associated with it is derived from stretching vibrations of the C = O bond of the amide, and it is a good predictor for quantitation of the protein secondary structure , regardless of the type of starch acetate. This might be due to the conversion of less ordered β‐sheet and β‐turn structures into α‐helices, which possessed the compact organization during reheating. During holding, water migration interrupted the organized hydrogen bonding system, and thus both covalent and noncovalent bonds began to break, leading to depolymerization and the decrease in the ratio of α‐helix and β‐sheet. As shown in Table The ratio of α‐helix and β‐sheet in the FCN sample with the addition of starch acetate was higher than that of control, which might be due to the better freezing thaw stability of starch itself after acetylation, causing less damage to the gluten network. A previous study found that the α‐helix structure was more hydrophobic, and increasing confirmation of α‐helix might lead to a more ordered structure. Thus, higher α‐helix content reflected greater water mobility and resulted in more dough stability ; Project administration (supporting). Di Zhao: Data curation (lead); Formal analysis (lead); Writing – original draft (lead); Writing – review & editing (lead). Dongxu Guo: Data curation . Xiaofeng Tong: Formal analysis . Yun Zhang: Formal analysis (supporting). Lingbo Qu: Supervision (supporting).


The swelling power decreased with a longer reaction time, reaching the lowest pH 10. In contrast, the carbonyl and carboxyl content exhibited the same pattern as solubility. A more alkaline environment tended to create modified starch with more favorable properties. Over time, oxidation shows more significant characteristics, indicating a superb product of this reaction. At the starch concentration of 20%, modified sago starch with the most favorable properties was created. When compared to modified starch, native starch is generally shaped in a more oval and irregular manner. Additionally, native starch and modified starch had similar spectral patterns and identical X-ray diffraction patterns. Meanwhile, oxidized starch had different gelatinization and retrogradation temperatures to those of the native starch.The disadvantageous properties of sago starch has limited its application in food and industrial processes. The properties of sago starch can be improved by changing its physicochemical and rheological characteristics. This study examined the influence of reaction time, acidity, and starch concentration on the oxidation of sago starch with ozone, a strong oxidant. Swelling, solubility, carbonyl, carboxyl, granule morphology, thermal profile, and functional groups are comprehensively observed parameters. With starch concentrations of 10–30% ( Starch, which is mainly composed of carbohydrates, is an essential daily nutrient source. Starch can be produced from various plant parts, such as the seeds of corn, rice, wheat, tubers of cassava, yams, and potato, and the stem of sago palm . Starch Metroxylone sagu Rottb) grows well in Southeast Asia’s tropical rain forests between 100 northern and southern latitudes [The sago palm . During oxidation, the hydroxyl groups are substituted with carboxyl (HO–C=O) and carbonyl (C=O) groups; an increase in the content of carboxyl and carbonyl, causes the degree of substitution to increase . The car3) naturally changes into oxygen (O2) quickly. It will not leave any residue in the product [Ozone, a strong oxidizing agent, is widely used in water and wastewater treatment, because it can remove odor, color, taste, and soluble particles in water or suspensions. It has many applications in food processing ,24,25,26 product . Ozone pChan et al. (2009) have successfully modified sago starch with ozone; however, they have not comprehensively studied the morphological properties, functional groups, and thermal properties of the modified sago starch . SeveralMetroxylon sagu). Ozone was produced with an Ozonator (Aquasuper model LZH-103A) Moro Jaya Teknik, Jakarta, Indonesia with an oxygen supply (99.60–99.9%) from Samator Aneka Gas Industri. Merck provided NaOH, potassium iodide, amylum, HCl, AgNO3, NH2OH, Cl, and phenolphthalein indicator.Alfurqan Tribinatama company supplied sago starch “Engke” , X-ray diffraction (XRD), Transform Infra-Red (FTIR), and differential scanning calorimetry (DSC) analyses.The swelling power of modified sago starch was determined using the method outlined by Chan et al. (2009) . One-graThe solubility of modified sago starch was determined using the method presented by Chan et al. (2009) . One-gra− content. The filtrate was tested for Cl− content using AgNO3. If there were Cl− in the slurry, its color would be turbid. Subsequently, 300 mL of aquadest was added to the slurry that was free of Cl−. The solution was heated in boiling water until gelatin formed; heating was then continued for another 15 min. The hot solution was titrated with 0.1 N NaOH using phenolphthalein as an indicator. The carboxyl chain test was repeated using native sago starch. The percentage of carboxyl was calculated, as follows.The modified sago starch’s carboxyl was determined using the method that was developed by . Three-gThe carbonyl of the modified sago starch was determined using the method outlined by Sangseethong et al. . Four-grAn analytical scanning electron microscope  with 2000× magnifications was used to observe the granule surfaces of native and modified sago starch. SEM is a microscopic electron method used to analyze a sample’s morphology. It produces a sample image by scanning the sample with focused light from an absorption electron that produces an electron beam at an accelerated voltage of 2–30 kV .The functional group of modified sago starch was observed using a Fourier Transform Infra-Red (FTIR) Shimadzu .A differential scanning calorimeter was used to determine the temperature profiles of native starch and modified sago starch . Scanning was carried out at a temperature range of 30–200 °C with a scanning speed of 10 °C/minute. An empty pan was used as a reference, and the data were analyzed by TA-60WS software .The analyses were repeated twice for each sample. The data obtained are shown as the mean value ± standard deviation (Mean ± SD). The data were analyzed using One-way analysis ANOVA. Subsequently, we conducted a post-hoc test with Duncan test at intervals of 95% confidence for further analysis of each of these variables.v/w), and with longer oxidation time. On the other hand, at starch concentrations of 10%, 20%, and 30% (v/w), the carbonyl percentage at pH 10 was higher than pH 7 and pH 4. Additionally, the average number of carbonyl groups obtained at starch concentrations of 10%, 20%, and 30% was not significantly different (p > 0.05), ranging from 0.091% to 0.154%. The modified starch had the highest carbonyl percentage at 0.154% with a starch concentration of 20%, reaction time of 25 min. and pH 10.The carbonyl group (DSC=O) content is expressed in the number of moles of carbonyl per mole of anhydroglucose unit . The effv/w). Additionally, at starch concentrations of 10%, 20%, and 30%, the carboxyl group content at pH 10 was higher than at pH 7 and 4. Additionally, the average number of carboxyl groups obtained at various starch concentrations was not significantly different (p > 0.05), ranging from 0.045% to 0.150%. At the starch concentration of 20%, the carboxyl group content reached the highest value at 0.150% at 25 min. and pH solution 10.The carboxyl group content of the modified starch was obtained titrimetrically and expressed in the number of carboxyl moles per mole of anhydroglucose units . The outSolubility is the number of starch molecules that are dissolved at a specific temperature; it is expressed as grams of dry residual weight per 100 g of dry sample weight ,29. The v/w). On the other hand, at starch concentrations of 10%, 20%, and 30%, the swelling power at pH 10 was smaller than pH 7 and 4. The average swelling power at starch concentrations 10%, 20%, and 30% was not significantly different (p > 0.05), ranging from 1.314% to 3.102%. At the starch concentration of 20%, oxidation time of 25 min., and pH 10, the swelling power had the smaller value at 1.314%. The swelling power of native starch is higher, reaching 3.150%. The swelling power of starch has decreased after oxidation due to reduced amylose content [The effect of reaction time, pH, and starch concentration on the swelling power was examined . A longe content . Amylope content .SEM was used to compare the morphology of the native and modified starch granules at various pH values and starch concentrations with an oxidation time of 25 min  in order−1. The bands at 3000–3750, 2700–3000, and 1600–1800 cm−1 were assigned to stretch O–H, stretch C–H, and bend water, respectively [−1 [−1 bands. Additionally, the bonding to the carbonyl group, C=O, displayed absorption in the region of 1680–1750 cm−1. The O–H bond is another type of bond that is important for identification; it absorbs at different positions, depending on the environment. This bond is very easily identified as acidic because it produces a wide signal in the area of 2500–3300 cm−1 [FTIR analysis was used to compare the absorption at specific wavelengths between the native starch and modified starch . The speectively . The benvely [−1 . The anh300 cm−1 ,42.−4 nm [Diffraction as a physical phenomenon involves electromagnetic waves at a specific wavelength from a specific metal; thus, elastic reflections can be detected by varying the angle of reflection. The analysis of the materials as the atom plans are placed at comparable distances to the X-ray lengths can be explained as the application of this phenomenon . Here, X−4 nm .The XRD patterns of the native and ozone-oxidized sago starches were compared . The oxiDifferential scanning calorimetry (DSC) is used to determine starch’s thermal behavior . The thev/w), and with an increased time of oxidation because more oxidants converted the hydroxyl groups into the carbonyl groups. Hydrogen bonds influence the integrity of the native starch grains. The decreasing of hydrogen bonds between the modified starch molecules occurs due to hydroxyl group replacement. Thus, the carbonyl and carboxyl group formation were considered to cause granular weakening [The carbonyl group content shown in eakening . The careakening . Chan eteakening . Anothereakening ,47. While at various starch concentrations, the carbonyl group content at pH 10 was higher than that at pH 7 and 4, likely because carbonyl groups formed quickly at pH 7–7.5 . Starch v/w), and longer oxidation time due to the increased change of hydroxyl groups into carboxyl groups, as shown in The number of carboxyl groups increased at pH 4, 7, and 10, the starch concentrations of 10%, 20%, and 30% , the carboxyl group content at pH 10 was higher than that at pH 7 and 4, because carboxyl groups are easily formed at a pH over 8.5 [Here, at various starch concentrations of 10%, 20%, and 30% (over 8.5 . At a pHover 8.5 . Therefoover 8.5 .v/w) and longer oxidation time. On the other hand, at starch concentrations of 10%, 20%, and 30%, solubility at pH 10 was higher than at pH 7 and 4. However, the average solubility at various starch concentrations was not significantly different (p > 0.05), ranging from 0.181% to 0.444%. At the starch concentration of 20%, oxidation time of 25 min., and pH 10, the solubility was highest, at 0.444%. In contrast, the solubility of the native starch was much lower, at 0.138%.The ozonation time, pH, and starch concentrations all appeared to affect the modified starch’s solubility . The solp > 0.05) [The solubility of modified sago starch is higher than native starch. The duration of starch oxidation increases starch solubility due to depolymerization and the weakening of the structure of the starch granule. The solubility of oxidized sago starch increased with an increasing moles number of oxidizing agents, but not significantly ( > 0.05) ,29. The  > 0.05) . Thus, i > 0.05) .Regarding the effect of pH on solubility, at pH 10, the modified starch had higher solubility than at pH 7 and 4, because, at pH 10, the number of carboxyl and carbonyl groups generated was higher than at pH 7 and 4. The increase in carboxyl and carbonyl groups causes starch granule structure to weaken, which increases the solubility. Thus, in general, starch solubility increases after oxidation. In this case, the carboxyl groups strongly affect the amylose degradation.Meanwhile, amylose molecules that degraded into short-chain molecules begin to dissolve in water, which increases the solubility of starch in water . The solThe swelling power of modified starch, or its ability to expand, is a property that characterizes the development of material. The contact between the starch granules and water causes the granule bodies to swell; thus, the swelling power indicates the ability of starch to hydrate under different conditions . The sweOverall, the value of swelling power decreases with an increasing oxidation time. The structural disintegration in starch granules during the modification process reduces swelling . AmyloseHere, oxidation did not significantly change the morphology of sago starch. Some of the sago starch granules underwent erosion during oxidation, and the surface became rough, with several cracks and pores appearing on the surface, while the edges partially disappeared into irregular shapes b–f. HoweAccording to Klein et al., pH greatly affects the surface morphology of starch granules . OxidatiThe O–H group of sago starch with a starch concentration of 20% and pH 10 had a higher value than the native starch. The increase of the percentage of transmittance demonstrates the weakening of the O–H bond in sago starch, which indicates the oxidation of O–H bonds to the carbonyl and carboxyl groups ,43. AddiThese results  indicateThe modification of sago starch was found to affect physicochemical properties. Oxidation with ozone significantly affected the solubility, swelling power, and carbonyl and carboxyl content of the native starch. Native starch is more oval-shaped and irregularly shaped than modified starch, according to SEM analysis. Additionally, in the FTIR analysis results, between native starch and modified starch changes in absorption at specific wavelengths, the spectral patterns of all the samples were similar. In addition, the oxidized sago starch showed identical XRD patterns to that of native starch. On the other hand, DSC analysis revealed that the profile of gelatinization and retrogradation temperatures of oxidized starches was insignificantly different from native starch.


The study investigated the effects of the addition of starch on the properties of oil palm biomass particleboard bonded with citric acid. Three kinds of oil palm biomasses were used in this study for the fabrication of particleboard, namely, oil palm frond (OPF), oil palm trunk (OPT), and empty fruit bunch (EFB) particles. Citric acid and tapioca starch at the mixing ratios of 100:0, 87.5:12.5, and 75:25 were prepared at a 60% solid content. A 30% resin content based on the oven-dried weight of the oil palm biomass particles was used. The sprayed particles were pre-dried at 80 °C for 12 h before being hot-pressed at 180 °C and 4 MPa pressure for 10 min. The physical and mechanical properties of the particleboard were evaluated. The mixtures of citric acid and tapioca starch were characterized by thermogravimetric analysis (TGA). Thermal stability of citric acid was reduced after the addition of tapioca starch. The addition of 12.5% tapioca starch improved the bending strength of the particleboard but increased the thickness swelling slightly. All UF-bonded particleboard exhibited significantly inferior performance than that of citric-acid-bonded particleboard. Citric-acid-bonded particleboard maintained its original shape after being subjected to a cyclic-aging treatment, while the UF-bonded particleboard disintegrated half way through the treatment. The performance of EFB particleboard was significantly inferior to its OPT and OPF counterparts. Malaysia is one of the main palm oil producers and exporters worldwide. As of December 2020, the total oil palm planted area in Malaysia amounted to 5.865 million hectares. Of the planted area, 53.3% is in East Malaysia (Sabah and Sarawak), while the remaining 46.7% is in Peninsular Malaysia ,2. AccorCitric acid is a natural chemical substance that could be found in various fruits and vegetables, especially in citrus fruits. Lemons and limes are among citrus fruits that contain a higher concentration of citric acid . Thanks Dendrocalamus sp.) particleboard bonded with different ratios of citric acid and starch. It was found that an addition of 12.5 wt% starch could improve the dimensional stability and bending strength of the particleboard. The authors attributed the improvement to the strong hydrogen bond interactions between citric acid and starch. The authors also reported that the amount of amylose in starch is a deciding factor to the dimensional stability of particleboard. Starch with higher amylose tends to result in particleboard with better dimensional stability as the linear chains of amylose are able to form strong linkages that are responsible for better water resistance. In the production of particleboard, many additives could be added to the citric acid in order to improve the performance of the resultant boards. For example, Umemura et al.  added suImperata cylindrica) [Arundo Donax L.) [A variety of wood and non-wood materials have been used in the production of citric-acid-bonded particleboard such as bamboo , teak 1, sweet sindrica) , new giaonax L.) , sugarcaonax L.) , and rubonax L.) . Howeverw/w).Three types of oil palm biomasses, namely, oil palm trunk (OPT), oil palm frond (OPF), and empty fruit bunch (EFB) were used in this study. Both OPT and OPF were collected from the felled oil palm trees in an oil palm field at Universiti Putra Malaysia. A portable wood chainsaw  was used to reduce the size of the OPT and OPF before ground into particle sizes. On the other hand, fibers of EFB were supplied by a local company located at Dengkil, Selangor. The distribution of the particles’ width and length are shown in 3 were produced. Three boards produced from a urea formaldehyde (UF) resin (65% solid content) was used as a control. Ten wt% resin loading was used to produce the control particleboard. One wt% ammonium chloride was added to the UF resin as a hardener. After hot pressing, the boards were conditioned in a conditioning room setting at a temperature of 20 ± 5 °C and a relative humidity of 65 ± 5% until a constant mass was attained. The conditioned particleboards were then trimmed and cut according to the dimension requirements stated in JIS A 5908:2003 [Single-layer particleboard was produced using citric acid and tapioca starch at different ratios . Thirty percent resin loading based on the oven-dried weight of oil palm biomass particles were sprayed on the particles. The mixture was then mixed at room temperature for 5 min using a mixer machine (custom made). The mixing process was carried out carefully to ensure the mixture of citric acid and tapioca starch were spread evenly with the oil palm biomass particles. The mixed particle was then oven-dried in an oven at 80 °C for 12 h prior to hot pressing. The oven-dried particles were then hand-formed into a mat using a 340 mm × 340 mm wood mold. The mat was first pre-pressed to compact and then hot-pressed at 180 °C for 10 min with a 4 MPa pressure using a 100 Ton hydraulic laboratory hot press . The pressing parameters were chosen according to the study by Lee et al. , where a−1) in the Laboratory of Biocomposite Technology, Institute of Tropical Forestry and Forest Products, Universiti Putra Malaysia, Serdang, Selangor. TGA of citric acid and topical starch mixture in powdered form was performed under a nitrogen atmosphere using the instrument TGA Q500 V20.13 Build 39  between 30 and 600 °C  standard. The determination of lignin of the samples was conducted in accordance to TAPPI standard T222 os-74 , while tTest specimens were trimmed and prepared according to JIS A 5908:2003 [Drying at 105 °C for 10 hImmersion in water at 70 °C for 10 hDrying at 105 °C for 10 hImmersion in boiling water for 4 hDrying at 105 °C for 10 hThe physical properties of the particleboard including water absorption (WA) and thickness swelling (TS) produced were evaluated in accordance with JIS A 5908:2003 . The immThe weight and thickness changes of the samples after every stage of the treatment were recorded. Five replicates were used for every test. p ≤ 0.05) was performed. Tukey’s honest significant difference (HSD) test was used to further determine the significant level of average values for each treatment. The data collected were analyzed using Statistical Analysis System (SAS) software . Analysis of variance (ANOVA) at a 95% confidence level  [3) [3) [As for bulk density, EFB had the highest bulk density of 670.20 kg/ml et al.  who repobagasse 1 kg/m3 [2/m3) [3) , and bam [3) [3) .80 (temperature for 80% weight loss), which was 388.3 °C, followed by 87.5:12.5 (281.4 °C) and pure citric acid (228.0 °C). The final residues were 13.7%, 7.6%, and 2.2%, respectively. This might be due to the crosslinking of citric acid with starch through covalent bonding. As a higher degree of crosslinking with citric acid was achieved at higher starch loadings, the final residue also increased correspondingly [The particleboard was hot-pressed at 180 °C in this study. At this temperature, pure citric acid experienced a weight loss of 5.9%, while the mixture of citric acid and tapioca starch at 87.5:12.5 and 75:25 recorded weight losses of 9.0% and 15.8%, respectively. The event implies a lower thermal stability of the mixture at a lower temperature. However, the mixture of citric acid and tapioca starch showed a higher resistance at a higher temperature, and their final residue was much higher than that of pure citric acid. Overall, the mixture of citric acid and tapioca starch at a ratio of 75:25 had the highest Tondingly . Additioondingly . 2 and 972.75 N/mm2, respectively. The MOR and MOE values increased as citric acid was used as a binder. It is interesting to note that the MOR of the OPF particleboard increased when higher starch was added to the citric acid. As for the MOE, the addition of 12.5% starch increased the MOE of the particleboard, but it started to decrease when 25% starch was added. On the other hand, OPT particleboard experienced the same trend as the OPF particleboard in terms of MOE. However, OPT particleboard showed a decreasing MOR trend as the starch addition increased. The MOR of OPT particleboard bonded with pure citric acid and 87.5CA:12.5 starch did not differ significantly. UF-bonded EFB particleboard had the lowest MOR value of 4.49 N/mm2. However, EFB bonded with citric acid exhibited a higher MOR, particularly those bonded with 87.5CA:12.5 starch, which has a statistically higher MOR value than UF-bonded EFB particleboard. The MOE values of the citric-acid-bonded EFB particleboard also displayed a decreasing trend with increasing starch addition, but no significant different was detected between particleboard bonded with pure citric acid and 87.5CA:12.5 starch. UF-bonded OPF particleboard had an MOR and an MOE of 6.89 N/mm2, 0.44 N/mm2, and 0.35 N/mm2, respectively. However, with the addition of 12.5% starch, the IB strength decreased around 11.5%, 10%, and 28.6% for OPF, OPT, and EFB particleboard, respectively. The IB strength of the particleboard experienced further decrement when 25% starch was added. All the particleboard bonded with citric acid achieved the minimum requirement of IB strength (0.15 N/mm2) for type 8 particleboard as specified in JIS A 5908. Overall, with the exception of some outliers, the addition of 12.5% starch was beneficial to the bending strength of the particleboard. Further starch addition (25%) could cause a disastrous effect to the mechanical strength of the fabricated particleboard.Citric-acid-bonded particleboard had a higher IB strength than that of UF-bonded particleboard. A very consistent trend was noticeable for the IB strength of citric-acid-bonded particleboard as the IB values decreased with increasing starch addition. The maximum IB strength of pure citric-acid-bonded OPF, OPT, and EFB particleboard were 0.52 N/mmAmong the oil palm biomasses, particleboard made from OPF particles seemed to have the highest mechanical properties. This may be due to fact that OPF possesses thick fiber walls. Better bonding could be achieved as the lumen to cell wall ratio is high in OPF, which means OPF particles could be easily compressed closely together . Both OPThickness swelling (TS) and water absorption (WA) of the particleboard fabricated in this study are listed in 2h of the UF-bonded OPF, OPT, and EFB particleboards were 48.07%, 44.19%, and 90.86%, respectively. Meanwhile, TS24h values were 71.22%, 59.96%, and 144.02%, respectively. The observation is unsurprising as UF resin is known to be very instable at high relative humidity owing to its susceptible aminomethylene linkage [24h values of OPF, OPT, and EFB particleboards bonded with pure citric acid (100:0) were approximately one-sixth, one-fifth, and one-seventh of their respective UF counterparts. Both OPF and EFB particleboard bonded with pure citric acid had significantly lower TS24h values than the particleboard bonded with starch addition. The findings were in agreement with Widyorini et al. [24h value of 7.28%. After 2 h immersion in water, the TS linkage . Howeveri et al.  who repo2h (46.97% to 93.29%) and WA24h (57.25% to 123.02%) than UF-bonded particleboard . The findings indicate that the citric acid inhibited water absorption by the particleboard during the water-immersion test [Similarly, particleboard bonded with citric acid had a significantly lower WAion test  stated tion test . TherefoThe thickness and weight changes of oil palm biomass particleboards during cyclic aging treatment are shown in As shown in UF-bonded OPT particleboards are the most dimensionally stable ones compared to that of the OPF and EFB particleboards. UF-bonded OPT particleboard experienced disintegration after the second drying stage at 105 °C, right after immersion in warm water at 70 °C for 10 h. Dissimilar to OPF particleboard, OPT particleboard bonded with 87.5CA:12.5 starch displayed the lowest thickness change of 11.69% after being boiled in hot water for 4 h a. The thFor EFB particleboard, UF-bonded particleboard experienced disintegration after the first drying stage at 105 °C, which was similar to UF-bonded OPF particleboard. The thickness change value of EFB particleboard bonded with pure citric acid was 35.08% after being boiled in hot water for 4 h a. MeanwhGenerally, in most of the cases, the thickness and weight change values increased along with increasing starch content. OPT particleboard was the only exception, where the particleboard bonded with 87.5CA:12.5 starch displayed the lowest thickness change. It can be concluded that the addition of starch contributed to the increment of water-soluble compounds in the particleboards. As a result, the water resistance of the adhesive reduced as the particleboards were subjected to repeated immersion cycles. The water penetrated into the particleboard and weakened the bond of the particleboard . Apart fWith the exception of UF-bonded particleboard, all the particleboard bonded with citric acid and the mixture of citric acid and tapioca starch maintained their original shape after cyclic-aging treatment . A darkeThe effects of the addition of starch on the properties of oil palm biomass particleboard bonded with citric acid were investigated in this study. Generally, the addition of starch reduced the thermal stability of citric acid when subjected to lower temperatures. However, the mixture of citric acid and tapioca starch showed higher resistance at higher temperatures as indicated by the higher amount of residues compared to that of pure citric acid. In terms of the physical and mechanical properties of the particleboard produced, an addition of 12.5% starch might be beneficial. All of the citric-acid-bonded particleboard showed significantly better performance than that of UF-bonded particleboard. Some of the particleboard displayed better bending strength when bonded with a mixture of citric acid and tapioca starch at ratio of 87.5:12.5. Although some particleboards showed a decreasing trend after an addition of 12.5% starch, the decrement was, however, insignificant compared to those bonded with pure citric acid. The addition of 25% starch was unfavorable as it resulted in an adverse effect on the mechanical properties of the particleboard. On the other hand, a similar observation was obtained for physical properties of the particleboard. The addition of 12.5% starch slightly increased the TS and WA of the particleboard  due to the solubility characteristic of starch in water. The increment was, however, acceptable. When subjected to the cyclic-aging treatment, citric-acid-bonded particleboard displayed superior dimensional stability as they maintained their original shape after the treatment. On the contrary, all the UF-bonded particleboard disintegrated half way through the treatment. Among oil palm biomasses, OPT and OPF exhibited better performance than that of EFB in terms of mechanical strength and dimensional stability. In conclusion, citric acid is a promising binder for particleboard as well as other wood composites. The addition of 12.5% starch could be beneficial to the mechanical properties, but addition beyond that loading is not recommended.


Numerous methods and devices are available for implant fixation in anterior cruciate ligament (ACL) reconstruction. Biomechanical data indicate high variability in fixation stability across different devices. This study aims to provide a better insight into measuring the structural characteristics and mechanical behavior of ACL implant fixations. Fourteen human tibial specimens with reconstructed ACLs were subjected to progressively increasing dynamic loading until failure. The motions of the tibia, the proximal and distal graft ends, as well as the testing frame and actuator, were continuously recorded via a motion tracking system. Significantly higher displacements of the machine actuator  were measured compared to the displacements of the proximal  and distal graft  ends. The displacements measured at different sites showed significant correlations. The provided data suggest significant and systematic inaccuracies in the stiffness and slippage of the fixation when using machine displacement, as commonly reported in the literature. The assessment of the distal graft displacement excludes the artifactual graft elongation, and most accurately reflects the graft slippage onset indicating clinical failure. Considering the high displacement at the ultimate load, the ultimate load could be used as a standardized variable to compare different fixation methods. However, the ultimate load alone is not sufficient to qualitatively describe fixation stability. The rupture of the anterior cruciate ligament (ACL) is among the most common knee injuries. Complete ruptures are typically treated surgically ,2. An ACFourteen anatomic specimens of human tibiae, with preserved semitendinosus and gracilis tendons, were obtained from 3 female and 8 male donors (age 72.5 ± 5.7 years (mean ± standard deviation), range 62–79 years). The fresh-frozen specimens were thawed at room temperature for 24 h prior to preparation. They were also kept moist with a 0.9% saline solution between the preparation steps, and tested at room temperature. The most proximal part of each tibia was cut at a total length of 110 mm using an oscillating saw, and carefully stripped of soft tissue. Following the surgical procedure (2.2), the distal 30 mm was embedded in polymethyl methacrylate  cylinders of 80 mm diameter and 28 mm height. The embedding was secured in the cup by means of twelve 5 mm screws with a conical tip. Projected line laser beams ensured that the graft tunnel and the graft were aligned with the vertical axis representing the biomechanical worst-case scenario.The semitendinosus and gracilis tendons were utilized in all specimens as ACL replacements. The tendon grafts were harvested in their full length, quadrupled and, if necessary, longitudinally trimmed to match the 8 mm diameter sizing sleeve. The distal 30 to 40 mm of the tendon grafts were then sutured in a whipstitch manner using No. 1 Polysorb suture , and pretensioned at 50 N. A guide wire was placed anatomically in the proximal tibia in lateral to the medial direction at an angle of 55°, followed by the incremental drilling of the bone tunnel diameter from 5 mm up to 8 mm. The graft was inserted into the distal end of the tunnel and pulled in proximal direction. A 1.0 mm guide wire was placed laterally at the interface between the tendon graft and the tunnel followed by the placement of a fully threaded 8 × 28 mm BioComposite Interference Screw , or an 8.0 mm Shark Screw ACL , designed for ACL fixation over the same guide wire. The presented samples are a subpopulation from a study evaluating the fixation stability of different fixation devices (data to be published).As visualized in S) were computed to investigate the strength of correlation between the displacement measured at the different test setup locations, at slippage onset and ultimate load. Machine and proximal graft end displacement, as well as graft strain, were considered dependent variables. The significance level was set to p = 0.05 for all statistical tests. Data obtained from the specimens with two different implant fixations were pooled. Values described in the text are mean and standard deviations (SD).For data analysis, displacements at two events, namely, at the onset of slippage and when reaching the ultimate load, were assessed from continuously recorded data. The statistical analysis was performed using the SPSS software package . The data of each outcome measure were tested for normality of distribution using the Shapiro-Wilk test. Given the non-normal distribution (NND) for most variables, the Wilcoxon signed rank test was performed to test for statistically significant differences between the outcomes measured at the different locations of the test setup. Monotonic regression analysis was performed, and Spearman’s correlation coefficients . The displacement at ultimate load, measured by the machine, was significantly higher than the displacements of both the proximal and distal graft ends (both p = 0.001). The proximal graft end displacement was significantly higher than the distal one (p = 0.009).At the slippage onset, there was no significant difference between the displacements of the proximal graft end and of the machine actuator  the significant difference between displacements measured at the proximal, as well as the distal graft end and the machine actuator, and (ii) the significant correlations between displacmenets measured at different sites. Commonly, stiffness is calculated from a linear portion of the load-displacement curve as the ratio of the applied load and the corresponding deformation. In the literature, the displacement of a fixation in ACL reconstruction has usually been represented by the displacement of the load frame, i.e., machine actuator displacement . Clinically, an additional concurrent slippage at the femoral side may be expected, leading to a higher degree of joint laxity. Therefore, the ultimate load alone may not be a sufficient clinically relevant indicator for fixation strength. On the basis of clinical investigations, biomechanical studies e.g., ,31) have have31])While the tendon graft strain remained relatively low (3% at ultimate load), it showed high deviations, reached maxima of up to 15%, and it did not correlate with the applied tensile load. This finding indicates that tendon graft strain cannot be assumed as a constant variable across all specimens. Graft strain has a specimen-specific effect on the measured deformation and, furthermore, on the system stiffness. When targeting the assessment of fixation stability, the variation caused by graft compliance has to be regarded as a confounding factor.In our study, the absolute displacement of the markers placed at the proximal end of the tibia in the direction of the applied load was a result of the possible relative displacement of the PMMA embedding in the load frame, the relative displacement of the bone in the embedding, or the elastic deformation of both components. However, all these displacements remained in the submillimeter range and were, compared to the displacement of the tendon and the machine actuator, negligibly small.in vivo conditions in biomechanical studies is difficult and deviates from simplified models with simulated pathologies. The presented data are based on experiments with two different fixation screws, while there is a wide range of fixation devices and methods available. Other systems might show different slippage onset resulting in a different effect magnitude. The authors have chosen to perform the investigation using fixation screws because interference screw fixation is a frequently applied technique [This study has limitations inherent to all biomechanical cadaveric investigations. In addition, it is limited by the high donor age, which does not fully reflect the typical patient cohort undergoing an ACL reconstruction. Furthermore, the applied unidirectional cyclic loading with increasing amplitude ,18,19,20In conclusion, the experimental assessment of the stability following implant fixation methods in ACL reconstruction relies on the accurate measurement of the relative displacements and deformations of the tendon graft. The use of machine actuator displacement overestimates the true slippage of the graft because of the compliances of various components. However, it allows a qualitative comparison between different fixation methods and devices. If feasible, displacement should be assessed at the distal graft end to exclude artifactual graft elongation. The ultimate load can be used as a standardized variable for the comparison between different fixation methods. However, the ultimate load alone is not a sufficient measure to qualitatively describe fixation stability.