Background: Giant gourami , belonging to gurami sago strain, is an important economic fish species that was newly released for domestication in 2018 in Indonesia. The present study aimed to determine the growth, production and feed conversion efficiency of gurami sago strain in different aquaculture systems.Methods: A mean of 240 juveniles were stocked  into concrete ponds, ﬂoating net cages and earthen freshwater ponds (12 m3) with three replicates of each. The juveniles were fed a floating commercial pellet diet containing 30% crude protein and 5% crude lipids. Feed was supplied at 3% of fish biomass per day throughout the 90 days of the experiment. The research was conducted in the area surrounding Lake Maninjau of Indonesia.Results: After 90 days, the mean weight of ﬁsh reared in concrete ponds was 166.86 g, floating net cages was 179.51 g and earthen freshwater ponds was 149.89 g. The mean final biomass was 37.64 kg for concrete ponds, 41.27 kg for floating net cages, and 33.72 kg for earthen freshwater ponds. The specific growth rates (%/day) for concrete ponds, floating net cages and earthen freshwater ponds were 0.67, 0.75 and 0.62, respectively. The feed conversion rates were 1.45 for concrete ponds, 1.30 for floating net cages and 1.87 for earthen freshwater ponds. The net yields (kg mˉ3) were 2.05 for concrete ponds, 2.27 for floating net cages, and 1.73 for earthen freshwater ponds. The exponents (b) of the length–weight relationship were calculated for concrete ponds (1.0146), floating net cages (1.2641), and earthen freshwater ponds (1.0056).Conclusion: The study showed that the growth performance, production and feed conversion efficiency of the gurami sago strain were the best found in floating net cages and considered a new aquaculture system in the future. To meet the demand for food from aquaculture production arises competition use natural resources, such as land and waterOsphronemus goramy Lacepède (1801) is one of the main freshwater commodities of economic importance. This species has been produced in small-scale farms for decades in Indonesia8. However, only contributed as much 6.96% of the total freshwater aquaculture production. Meanwhile,Nile tilapia,Clarias catfish,Pangasius catfish, and common carp has been contributed 37.93%, 33.35%, 12.38%, and 9.28% of 3,374,924 metric tons freshwater fish production9. Therefore, there are still important gaps of knowledge in its aquaculture of giant gourami.The giant gourami8. The giant gourami belongs to the local gurami sago strain has never been cultured intensively. This species is the result of newly released domestication in 201810, which still limited in West Sumatera Province of Indonesia11. Gurami sago is an herbivorous species which can consume a variety of plants such as sente leaves (Alocasia macrorrhiza), kale (Brassica oleracea), cassava leaves (Manihot esculenta), and others young land plants. In addition, this species can eat commercial pellets, and tolerate crowded aquaculture production systems, such as earthen freshwater ponds and artificial ponds lined with membranes12.Although the contribution from giant gourami was lowest (6.96%), but the local gurami tambago and gurami galunggung strain have been cultured in semi-intensive11. This species grows well in nursery ponds and reach a market size of 200 to 300 g per fish and a size of 50 to 100 g per fish as ornamental fishes. This characteristic creates commercial interest as a new species in an effort to develop freshwater fish farming in the future. Concrete ponds and floating net cages were options in the development of gurami sago culture. Many studies have found that continuous water flow systems in concrete ponds, artificial ponds lined with membranes, tanks, canvas tanks, pens and many other systems could be an alternative for fish aquaculture because these systems provide a high degree of control that can allow for high production16.The gurami sago strain has been detected as candidate species for production in middle-scale farm in Indonesia18. High production can be achieved at a low cost20. Fish farming in cages can achieve maximum growth with a high survival rate22. However, cage fish farming has advantages and disadvantages that must be considered before choosing a production system. The main disadvantages of fish farming in the floating net cages of lakes are that they are not ideal for land use and may cause massive fish deaths24. Meanwhile, the advantages of floating net cage aquaculture include high water circulation, solid waste not accumulating near cages, low water quality variation, and no electrical power required for water aeration26.In the last decade, cage systems have received more attention from both researchers and producers. Fish farming in cages can be practiced intensively27, giant gourami in earthen freshwater ponds and artificial ponds lined with membranes12.Nile tilapia in the ponds and cages28, and golden pompano in the floating cages29. Because the rearing of the gurami sago strain is relatively new, there are no parameters or best methods available to predict the growth performance, survival and feed conversion efficiency in a commercial rearing system. Therefore, knowledge about the contribution of gurami sago to each aquaculture system is very important to analyze. The current study was conducted to assess the growth performance, production, economic food conversion rate and waste load of feed of gurami sago strains in different aquaculture systems namely, concrete ponds, floating net cages and earthen freshwater ponds.Fish production systems in many countries use a variety of methods, e.g., carp in earthen freshwater pondsO.goramy) strain in this study in concrete ponds, floating net cages and earthen freshwater ponds in the area surrounding Lake Maninjau of West Sumatera Province of Indonesia. The study was founded by LPPM (Research and Community Service) University of Bung Hatta under the Indonesia Endowment Fund for Education, Ministry of Finance, Republic of Indonesia, through the competitive grants scheme called the Productive Innovative Research (Policy/Governance) 2019 with the contract number PRJ-99/LPDP/2019. This grant included ethical approval and permits to collect fish samples including permission to rear this species. The animals used in this study did not suffer during the experiment. Gurami sago was transported to concrete ponds, floating net cages and earthen freshwater ponds for rearing for 90 days, fed commercial pellets and measured for growth performance every 30 days. At the end of the experiment, the gurami sago were still in good condition.There are no required permits from the government of the Republic of Indonesia to culture the gurami sago . The surface of the floating net cages was covered with nets stretched (25 mm mesh) to avoid bird predators.Each floating net cage has a size 4 × 2 m, a depth of 1.5 m and a volume of 12 m3. It had 50 mm of central drainage and was covered with a net of 0.5 cm mesh to prevent fish jumping and predator entry during the rearing activity. The water was pumped from wells at a velocity of 5 litres per minute.Each earthen freshwater pond has a size of 4 × 2 m, a depth of 1.5 m and a volume of 12 m3 (three replicates) were stocked with 240 juveniles each, with a density of approximately 20 fish/m3. The average initial weights and lengths of juveniles were 54.51±0.45 g and 13.81±0.02 cm (mean ± SD), respectively. The length was measured using a ruler with an accuracy level of 0.1 cm. The weight of each individual was measured with an electronic balance .The experiment ran for 90 days beginning on 01 April and ending on 29 June 2019. Approximately 3,000 gurami sago juveniles weighing approximately 50 g were obtained from a hatchery in the Luhak sub-district in the district of Lima Puluh Kota. Fish were acclimatized with 1000 juveniles per each pond . Fish were acclimatized to the floating net cages (5 × 5 × 3 m) for one month prior to the experiment. In the initial growth phase, three concrete ponds, three floating net cages and three earthen freshwater ponds of 12 m18. The amount of feed provided was as much as 3% per day based on fish biomass during the experiment. Every 30 days, samples were taken from ponds to monitor fish growth and to adjust the feed amount. Twenty-four fish samples were obtained from each concrete pond, floating net cage and earthen freshwater pond. 10% of the fish were sampled every month for each aquaculture system, due to giant gourami is sensitive to handling. Fish were captured at 07.00 AM with gillnets, which have a net bag with a suitable mesh size. Then, fish were anaesthetized orally with tricaine methanesulfonate , based on the dosage used forHemibagrus wyckii30.Fish were fed twice daily (09:00 AM and 17:00 PM) with commercial floating pellet feed ⍛C) and dissolved oxygen  were measured with an oxygen metre (YSI model 85). The pH values were determined using a pH metre  Co., Ltd, Thailand). The levels of ammonia , nitrite-nitrogen , nitrate-nitrogen , chemical oxygen demand , biological oxygen demand , alkalinity (mg Lˉ1), hardness (mg Lˉ1), total dissolved solids  and total suspended solids  were measured in each aquaculture system with replication according to standard procedures31. The nets of the floating cages were cleaned routinely to maintain water circulation in the fish rearing areas. The walls of the floating net cages were cleaned by divers in the water.Water parameters were recorded weekly in the concrete ponds, floating net cages and earthen freshwater ponds. The water temperature  or (Wt-Wi)/t, whereWt = final weight,Wi = initial weight, andt = time (day);Absolute growth rate  = (lnW1-lnW2/t × 100)Specific growth rate  = total number of fish at harvest × average final weight/cage capacityGross yield (kg mˉ3) = (harvested biomass - stocked biomass/cage capacity)Net yield (kg mFeed conversion efficiency (FCE) = [fish weight gain (g)/total feed ingested (g)]Apparent feed conversion rate (AFCR) = supplied feed/increase fish weightEconomic AFCR = cost/kg of fish weight × feed costWaste load of feed = [feed intake (kg)] × [waste load/kg of feed]The gurami sago were reared for 90 days, and the survival rate was estimated by checking the aquaculture systems every day and recording the results. Dead fish were removed immediately. The survival rate percentage was calculated by subtracting the number of dead fish from the initial number of the stock. The parameters were analyzed according to Aryanib,W = aL whereW is the total wet weight (g),L is the total length (cm) anda andb are variables of the length–weight relationships (LWRs) equations. These variables were estimated by the least square regression method. A t-test was used for comparison of theb values obtained in the linear regressions with the isometric value by equation33:st = (b – 3)/bS, wherest is the t-test value,b is the slope andbS is the standard error of the slope (b). The comparison of the obtained values of the t-test with the respective table critical values allowed for the determination of whether theb values were statistically significant as well as their inclusion in the isometric range (b=3) or allometric range . The degree of correlation between the variables was computed to determine the coefficient, R2. Fulton’s condition index was calculated as3)K=100 and final total weight (g) were used to determine the relationship of34. The 95% confidence level (p<0.05) was considered as the threshold to identify significant differences. All means are given with ± standard deviation (±SD). The canonical discriminant functions were used to analyze the water quality grouping between rearing systems.The data were analyzed using SPSS software . Kolmogorov-Smirnov statistics were used to test data normality. Then, Levine’s test was used to analyse the absolute residuals from homogeneity. One-way ANOVA was used to analyze the effect of each treatment, followed by post hoc Duncan’s multiple range testsˉ3), net yield (kg mˉ3), absolute growth rate (g dayˉ1), specific growth rate (% dayˉ1), AFCR, and economic food conversion rate (US$/kg gain) after 90 days of culture  on the mean final body weight (g), final biomass (kg), weight gain (g), gross yield  affected by the different rearing systems. A summary of the FCR, FCE and waste load feed from the five aquaculture species is presented inDuring the 90 days of the experiment, the gurami sago reared in floating net cages grew faster than those reared in concrete ponds and earthen freshwater ponds . At the W = 60.875et0.0498 (withR2 = 0.83) for the concrete pond,W = 48.580et0.0613 (withR2 = 0.75) for the floating net cage andW = 55.7050et0.0623 (withR2 = 0.75) for the earthen freshwater pond. The length–weight relationships for the gurami sago reared in concrete ponds were shown byW = 7.9368L1.0146  indicating negative allometric growth. The Fulton’s condition index in the concrete pond, floating net cages and earthen freshwater pond were 2.45, 1.91, and 3.36, respectively.The growth rates of gurami sago based on body weight were described according to the following exponential equation:In this study, the water quality was recorded weekly from each aquaculture system during the experiment period and showed significant differences (p<0.05) in terms of TDS, TSS, DO, COD, BOD, ammonia, nitrites, nitrates, pH, alkalinity and hardness, only water temperature did not show a significant difference. Furthermore, in the principal component analysis, PC1 accounted for 66.67% of the 12 parameters of water quality, which had a positive correlation with all water quality parameters. This result shows that value has an effect on the water quality parameters in aquaculture systems. Alkalinity, hardness, pH, and dissolved oxygen make high contributions to the aquaculture system . The plo39. In fact, the diversification of aquaculture systems with the efficient use of land resources can increase aquaculture production40. The comparisons between concrete ponds, floating net cages and earthen freshwater ponds are relevant to determine their relative per unit volume performance of juveniles-rearing of gurami sago. The rearing of gurami sago is an alternative diversity of aquaculture that can contribute to the development of commercial production in the future.The aquaculture industry needs environmentally friendly aquatic ecosystems. Therefore, aquaculture practices must use aquaculture systems that minimize waste loads and increase added value8 and gurami sago in the artificial ponds lined with membranes12. On the other hand, the survival rates of gurami sago in earthen freshwater ponds (89.44%) were higher than those of carps (65.74%) and stinging catfish (69.00%) in freshwater ponds27.Gurami sago was successfully reared in concrete ponds, floating net cages and earthen freshwater ponds. However, their growth performance was best in the floating net cages. The high survival rate of gurami sago was found in the floating net cages, which was similar to the gurami tambago strain-1) values of 0.67, 0.75 and 0.62, respectively. In contrast, Budiet al.41 stated that giant gourami belonging to the local gurami soang strain in the laboratory with initial weight of 15.83 g had faster growth with an SGR value of 2.13% dayˉ1. The specific growth rate of fish seems to be influenced by the initial weight, strains and aquaculture systems. The economic AFCR value of fish fed in floating net cages was lower than that of fish fed in concrete ponds and earthen ponds. Therefore, it can reduce the cost of feed and increase the economic benefits to producers. This condition indicates that the culture of gurami sago in floating net cages gives fish a chance to consume more feed. However, this AFCR was lower than that ofNile tilapia43, and giant gourami8, and higher than the African catfish AFCR value44.The growth rate of gurami sago, with an average initial weight of 54.18 g, was faster in floating net cages than in concrete ponds and earthen freshwater ponds, with specific growth rate 8, 25.4 to 26.3 kg mˉ3 for pirarucu (Arapaima gigas)49, 88.5 kg mˉ3 for silver perch, (Bidyanus bidyanus)50 and 11.60 to 16.03 kg mˉ3 for spotted rose snapper (Lutjanus guttatus)36. It seems that different levels of aquaculture production can be influenced by species diversity, stocking density and duration of aquaculture. Giant gourami can produce a maximum profit after 324 days of aquaculture51.The aquaculture system influences the production of gurami sago. The highest production was found in the floating net cages, with a value of 3.36 kg met al,52 fish reared in cages can increase the variation in weight production. In contrast, the freshwater cages have been marred by increasing the frequencies of fish mortality, causing negative implications to finances and the environment53. Bosma and Verdegem54 reported that the direct risks related to aquaculture in ponds were habitat destruction, suboptimal freshwater consumption, organic pollution, eutrophication, and water contamination with pesticides. These factors can cause production to decline and cause low economic value.Herein, we recommend gurami sago strain aquaculture in concrete ponds, floating net cages and earthen freshwater ponds for 324 days. According to De Oliveira Continho55, the allometric coefficient (b) - calculated was 1.0146 for concrete ponds, 1.2641 for floating net cages and 1.0056 for earthen freshwater ponds. Gurami sago grown in different aquaculture systems showed negative allometric growth. These values were smaller than 2.94 for the culture ofTilapia zillii56 and 2.99 and 2.93 forPangasianodon hypophthalmus andClarias gariepinus, respectively57. The K-values were not different among concrete ponds, floating net cages and earthen freshwater ponds. The finding explains that no different morphological factors were found in gurami sago cultures in concrete ponds, floating net cages and earthen freshwater ponds. However, cultures of gurami sago in floating net cages had a smaller condition factor or had values close to 1.00. The variation in the condition factor (K) of gurami sago may be influenced by different factors, such as environmental conditions, feed intake and increased of body weight. The condition factor (K) of fish depends on many factors, including species diversity, growth, physiological performance, age, and gonadal maturity60.In this study, after the analysis of growth performance and production, we also analyzed the length–weight relationship and condition factor (K) from aquaculture systems. The exponent of the length–weight relationship - or per FroeseIn conclusion, our study showed that gurami sago strain can be efficiently reared in concrete ponds, earthen freshwater ponds and floating net cages. For all tested parameters, the best aquaculture system was found in the floating net cages. Nevertheless, further investigations on fish farming in the floating net cages which a technically feasible and economics at a larger scale are needed to determine commercial interest and environment impacts, especially on water quality, in an effort to develop of gurami sago fish farming in Indonesia.https://doi.org/10.6084/m9.figshare.11719542.v161.Figshare: Row data growth performance of gurami sago in different aquaculture systems.doc,– N=24)Table 1. Sample size of weight and length of the gurami sago strain  in the concrete pond culture (– N=24)Table 2. Sample size of weight and length of the gurami sago strain  in the floating net cage culture (– N=24)Table 3. Sample size of weight and length of the gurami sago strain  in the earthen freshwater pond culture (– N=24)Table 4. Sample size means of initial weight, final body weight and weight gain of gurami sago (– N=24)Table 5. Sample size means of initial length, final total length and length increase of gurami sago (– N=24)Table 6. Data on mean initial biomass, final biomass and gross yield of gurami sago (– N=24)Table 7. Data on mean SGR, feed intake and apparent feed conversion rate of gurami sago (– N=24)Table 8. Data on mean economic food conversion, feed conversion efficiency and waste load of feed (– N=24)Table 9. Data on mean growth (g) of gurami sago at 0 days, 30 days, 60 days, and 90 days (– N=72).Table 10. Data on mean size distribution (g) of gurami sago in the different aquaculture systems in the 90-day trial  of gurami sago_12 Feb 2020.doc,Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the General comment: I carefully read this revised version of the manuscript and, unless I am mistaken, none of my previous comments have still be considered. No rebuttal and/or answer has been provided as well. So, for these reasons, I still have some reservations about the publication of this, moreover very interesting, study. I hope my previous comments would help the Authors to revise their manuscript.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:aquaculture, aquatic ecotoxicologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I have read and compared between version 1 and version 2, the article has some improvement. There are some points that should be taking into attention for the authors for the next research and I can conclude that this article can be indexed after an amendment.ABSTRACTMethods: A mean of 240 juveniles were stocked , please add the word .Conclusion: Please mentioned which production is better among different aquaculture systems.INTRODUCTIONSufficient.MATERIALS AND METHODSSufficient.RESULTS AND DISCUSSIONWell written and organized.CONCLUSIONAs mentioned in the summary: “our study showed that gurami sago strain can be efficiently reared in floating net cage”, in which term gurami can be efficient rear in floating net cage for all parameters or some? Please add information more briefly.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Fish nutrition, Animal Physiology, Biology MolecularI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. We have been added some information to complete our article :Response for Rudy Agung Nugroho Comments:ABSTRACT : methods: We have been added the word initial weight means, 54.53 g and initial length 13.88 cm in the sentenceConclusion : The best aquaculture system was found in the floating net cages.CONCLUSION : In conclusion, our study showed that gurami sago strain can be efficiently reared in concrete ponds, earthen freshwater ponds and floating net cages. For all tested parameters, the best aquaculture system was found in the floating net cages. The desired corrections have been addressed. There are still some grammatical issues in the text, but I presume the editorial staff will handle this.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquaculture nutrition and bioenergeticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I acknowledge the Authors did revise their manuscript. Nevertheless, most of my previous comments remained not addressed in the revised version. The Authors should argue why they did not consider these comments. Please find below the comments, listed by sections, that should be addressed before submission of a new revised version.General comment: I believe it is really important at this point that the Authors deeply revise Introduction and Materials and Methods taking into account all the comments of the reviewers to ensure that the objectives of the study are clear, that the experience is scientifically valid and that the data analysis has been performed correctly. This is why I am waiting for a thoroughly revised version before providing additional comments on the Results and Discussion.Introduction: The flow of the Introduction may be improved. Thus, the first paragraph is not informative enough and too general. I suggest providing relevant figures about freshwater finfish production in Indonesia showing that this country is among the main producing country in the world. Some sentences could be added regarding the important role of freshwater aquaculture in the country by providing food and economic resources as well as employment in rural communities… Then the second paragraph should be focused on giant gourami as Authors did but maybe in a awkward way because the sentences said that giant gourami is important but this species accounts for only 7% of the national production. Even if it is true, giant gourami production represents now more than 100,000 tonnes/year of production and this production is growing (latest available production volumes and increasing rate per year should be provided). This species has high patrimonial and economic values (among the most expensive freshwater fish species in Indonesia). Instead of having the comparison of production volumes with other freshwater fish species, I suggest to the Authors to provide data and relevant arguments to highlight the importance of giant gourami in Indonesia. Most of the information should be available in the following references: FAO (2019) Cultured Aquatic Species Information Programme. Osphronemus goramy. Cultured AquaticSpecies Information Programme. Text by Caruso, D., Arifin, Z.O., Subagja, J., Jacques Slembrouck, J.and New, M. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 26 September 2019 FAO (2020). FishStatJ: software for fishery statistical time series. FAO, Roma. Thus, the structure of the Introduction could be, with appropriate transition sentences: 1/ Importance of freshwater finfish aquaculture in Indonesia 2/ Giant gourami among the main freshwater commodities in the country 3/ Gaps of knowledge in gourami aquaculture 4/ Rearing structures for giant gourami aquaculture 5/ Objectives of the studyOsphronemy goramy. Here again, as I said on my previous report, I do not see the point to be focused on this strain in the Introduction because the findings from this study may be interesting for giant gourami in general. The strain used should be only mentioned in the Materials and Methods section. In my previous report I highlighted the confusion between “strain” and “species” with those terms used interchangeably throughout the manuscript. Again, gurami sago is not a new species as the Authors said, it is a strain of giant gouramiMaterials and Methods: In the subsection “Ethical considerations”, I think the sentence “The animals used in this study did not suffer during the experiment” should be deleted because it is always difficult to assess suffering in fish. The most important information regarding ethical considerations was already provided. As said in the previous review performed by Reviewer 2, water parameter values should be included in a Table within the manuscript, not as additional data. Change “Sampling design” by “Experimental procedures” In this subsection, when the origin of the fish is provided I suggest saying that the fish belonged to the “Gurame sago” strain, moving the information regarding this strain from the Introduction. I suggest rephrasing the following sentence “Twenty-four fish samples were obtained from each concrete pond, floating net cage and earthen freshwater pond. 10% of the fish were sampled every month for each aquaculture system, due to giant gourami is sensitive to handling.” to “Twenty-four fish were sampled monthly from each concrete pond, floating net cage and earthen freshwater pond. The sample size and sampling frequency were selected in order to sample 10% of the total number of fish for each rearing structure while limited stress for the fish caused by handling.” I am not sure to understand how the “Waste load of feed” and “FCE” were evaluated since they require to be able to quantify the feed ingestion, right? I do not know how such an estimation can be performed in the tested rearing systems. The Authors should clarify this point already highlighted in the previous report. For the data analysis, assumptions of ANOVA  should be tested and met on residuals, it should be clearly indicated in the manuscript. “Levine’s test” should be corrected by “Levene’s test”. “One-way ANOVA was used to analyze the effect of each treatment”, effects on what? The Authors should be precise regarding the data analyzed through ANOVA. I guess all of them did not meet the assumptions for using parametric ANOVA. In the same section, the Authors said they used discriminant analysis to examine grouping of the rearing systems using water quality parameters but the terms “Principal Component Analysis (PCA)” were used in their response to the reviewer comments that are not referring to the same statistical approach. The actual analysis performed should be clarified and better described in the data analysis section by explaining potential data transformation prior to the analysis, how the principal components have been selected…Figures and Tables As I said in my previous report, the Authors should provide visible standard deviations values and statistical differences in the Figures when it is appropriate and better axis scale in order to improve data readability. For allometric relationships, p-values for model estimates should be provided. Overall, the Authors should pay special attention to provide revised Figures allowing them to clearly visualize each data. Again, water quality parameter values should be provided in a Table within the manuscript.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:aquaculture, aquatic ecotoxicologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Introduction:1–3. Many studies state that aquaculture production depends on many factors, including its species, aquaculture system, technical efficiency, production inputs, and infrastructure” - This is a rather convoluted paragraph. Please rephrase to improve coherence.“Aquaculture activities are responsible for the supply of fish for human consumption. To meet the demand for food from aquaculture production, competition uses natural resources, such as land and water“In 2018, the total aquaculture production in Indonesia was 16,032,122 metric tons” - That is probably correct, but then you go on to state that it all comes from fish culture. I think (supported by FAO 2018 yearbook) that around 12 million tons is seaweed. Please check and correct.”synthetic sheet ponds..” - I would prefer “artificial ponds lined  with membranes” or something similarIt is not clear to me what is meant by ”middle-scale commercial culture”. Is this in relation to intensity? Please clarify.M&M:“Feed conversion efficiency (FCE) = [fish weight gain (g)/total feed ingested (g)] Apparent feed conversion rate (AFCR) = supplied feed/increase fish weight” - It is interesting that you have two indicators of feed performance where one is listed as apparent. I presume that feed waste was not collected in any of your rearing systems, and therefore all of your feed intake are apparent and based on  “supplied feed”, also the FCE.“Waste load of feed = [feed intake (kg)] – [final biomass (kg)]” - I am not familiar with this variable, nor do I completely understand what it signifies, but presumably, it should be biomass gain, and not just final biomass?Results:One of the things that can explain the observed differences in growth performance is likely to be your water quality parameters. While it is fine with the PCA plot, I would really like to see the water quality measurements in a table. Once these are available, perhaps it would be possible to discuss which water quality parameters would be essential to control to successfully produce gourami in land-based systems.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:Aquaculture nutrition and bioenergeticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Introduction:Introduction in first paragraph have been rephrase to improve coherences in the paragraph.We have omitted the data of Indonesian fisheries production in the introduction.“Synthetic sheet ponds” have been change to “artificial ponds lined with membranes”.“middle-scale commercial culture” meant “middle-scale farms”M & Met al. 2017.We used the “Feed Conversion Efficiency (FCE)” Formula based on Chatvijitkul Reference: Chatvijitkul, S., Boyd C.E., Davis, D.A., McNevin, A.A. (2017). Pollution potential indicators for feed-based fish and shrimp culture. Aquaculture 477: 43-49.We have been revised the formula of “Waste load of feed”. Please see in sub-bab “Measurement Parameters”.Results: In Table 1, the parameters of waste load /kg of feed were added The water quality parameters have been revised. We have data of water quality parameter in Table form, and it is has been state in raw data which have uploaded in Figshare (see Table 11 revised). Osphronemus goramy Lacepède, 1801) strain in different aquaculture systems" examines the zootechnical performances of a giant gourami Osphronemus goramy strain called “gurami sago” in three different rearing structures: concrete ponds, floating net cages and earthen freshwater ponds.The present manuscript entitled "Growth, production and feed conversion performance of the gurami sago . Considering the variations of most of the measured parameters that can be occur in rearing structures such as shallow earthen ponds sometimes on the same day, I think such analysis is not appropriate. Furthermore, Authors should state why only 10% of the fish were sampled every month. I guess is because giant gourami is sensitive to handling but this information may be interesting to add.Results: As I already mentioned, I have some doubts regarding the validity of the CDF using water quality data. I believe that the presentation of the results can be improved. Authors should provide visible standard deviations values and statistical differences in the Figures when it is appropriate and better axis scale in order to improve data readability. For allometric relationships, p-values for model estimates should be provided. Table 1: “Final food conversion rate (Fulton’s K)”, I guess it should be changed by “Condition factor (Fulton’s K)”Discussion: Although water quality is likely a key parameter to explain some of the observed differences in zootechnical performances among the rearing structures, unfortunately, since water parameter values were recorded only once a month, I think that there is not enough information provided to use these results.Conclusion: In the concluding paragraph, I expected clear recommendations for giant gourami aquaculture based on the findings from this study.References: I believe that some references relating to the aquaculture of the giant gourami are missing. I suggest to consider the following references which can be useful in the Introduction and Discussion: FAO (2019) Cultured Aquatic Species Information Programme. Osphronemus goramy. Cultured Aquatic Species Information Programme. Text by Caruso, D., Arifin, Z.O., Subagja, J., Jacques Slembrouck, J. and New, M. In: FAO Fisheries and Aquaculture Department [online]. Rome. Updated 26 September 2019.1 Arifin O.Z., Slembrouck J., Subagja J., Pouil S.,  Yani A., Asependi A., Kristanto A.H., Legendre M. (2020). New insights into giant gourami (Osphronemus goramy) reproductive biology and egg production control. Aquaculture 519: 734743.2 Kristanto A.H., Slembrouck J., Subagja J.,Pouil S., Arifin O.Z., Prakoso V.A., Legendre M. (2020). Egg and fry production of giant gourami (Osphronemus goramy): Rearing practices and recommendations for future research. Journal of the World Aquaculture Society 51: 119-138.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?PartlyAre sufficient details of methods and analysis provided to allow replication by others?PartlyReviewer Expertise:aquaculture, aquatic ecotoxicologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Introduction:The introduction has been improving. The paragraph go straight to the point of why giant gourami important in Indonesian Aquaculture.Methods:Overall, the method has been revised based on your comment. We also add water quality data which come from our daily logbook. Actually water quality parameters were recorded weekly, but at the first we show in the Table 3 only per month for results Principal Component Analysis (PCA). Now we have changed it into weekly recorded (Table 3).Results:The results have been revised based on your comment. The allometric relationships  have been added in Figures 3, 4 and 5. Furthermore, Table 1 has been revised.Discussion:The water quality parameters have been revised based on your comment. We have changed it into weekly recorded. We have data from our daily logbook which already recorded during the research period. The complete raw data shows in Figshare (Table 11 revised).ConclusionWe have been revised the conclusion.References:We have been added some references based on your suggestion.


Most Chinese migrants have been faced with obstacles to getting access to local public health services. Young migrants among internal migrants make a major contribution to the economy. However, the establishment of their health records has been ignored. This research was aimed at exploring the status and associated factors of the establishment of health records among young Chinese migrants.Data were obtained from the 2017 China Migrants Dynamic Survey (CMDS). Chi-square test and binary logistic regression were performed to investigate the associated factors of the establishment of health records among young Chinese migrants.Approximately 30.2% of young migrants had their health records established in inflow communities. Urban residence, medical insurance (insured), and lower average monthly household income were attributed to the establishment of more health records. Young migrants who were in northeast China and across provinces and immigrated for working or engaging in trade were less likely to have health records established. Young migrants who participated in social activities and public affairs activities and took type of people in touch as natives in the inflow area showed a higher possibility of establishing health records. Meanwhile, receiving health education and hearing about national basic public health services (BPHSs) were beneficial for establishing the health records of more young migrants.This study showed that the health records of young migrants had a relatively low establishment rate. Sociodemographic and migration characteristics, health status, public health services, and social integration factors were all related to the health record establishment of young Chinese migrants. Changes have taken place in urban and rural population structures due to the current construction of new urbanization. Migration is the core issue of the recent urbanization process of China . The 201“Equalization of Essential Public Health Services” is a rare national program that covers the prevention and control of non-communicable diseases (NCDs) for internal migrants . The latIn response to the right and health protection of migrants, “Healthy China 2030” explicitly proposes to promote the equalization of BPHSs to gradually narrow the differences in basic health services and the inequality of health between populations . “13th FAs highlighted above, it is of importance to promote the above-mentioned national program and establish the health records of more young migrants. However, data about the situation of young migrants are minimal because most of the research has focused on the migrants who are ordinary and elderly and suffer from chronic diseases , 14, 15.Data were retrieved from the 2017 China Migrants Dynamic Survey (CMDS), a cross-sectional and nationally representative survey organized by the National Health Commission of the People’s Republic of China . A totalThe subjects of this study are young migrants. The World Health Organization limits the age of young people to 44 . Young mThe establishment status of the health records of young migrants was measured by the survey question “Have you established your health records in your community?” The establishment of health records was coded as “yes=1” if the reply was “Yes, established”, and “no=0” if the answer was “No, I’ve never heard of it”, “No, but I’ve heard it” or “I don't know about it” , 21.Sociodemographic characteristics included gender , age (15–29 or 30–44), ethnicity (Han or minority), marital status , or married (first or non-first marriage)), Hukou status, education level (primary school or below or middle/high school/college degree or above), medical insurance (uninsured/insured), and average monthly household income (below 3000/3001–5000/above 5001 yuan).Migration characteristics were measured through five migrant-related variables, namely sample area , extent  and duration of migration (≤ 1 year/2–4 years/5–9 years/≥ 10 years), reasons for migration (working or engaging in trade/following the migration of family members/marriage-related/else) as well as settlement willingness (no/yes).Health status and public health services factors included self-rated health , hypertension or type 2 diabetes (no/yes), distance of medical facilities (≤15 min/> 15 min), hearing about national BPHSs (no/yes) and health education (no/yes). The status of health education was measured by the survey question “Did you receive health education in your community last year?” whose answers have the following nine items: Occupational disease prevention, sexually transmitted disease (STD)/acquired immune deficiency syndrome (AIDS) prevention, reproductive health and contraception, tuberculosis prevention, smoking control, mental health, chronic disease prevention, maternal and child health care as well as self-help in public emergencies. Health education was coded as “no=0” if none of the above items were selected and otherwise “yes=1.”According to existing studies and available data –24, fourp value less than 0.05 was considered as statistically significant. Before conducting binary logistic regression, independent variables were tested for co-linearity according to tolerance values and the VIF (variance inflation factor). The criteria values for tolerance and VIF  to identify co-linearity [Statistical analyses were carried out by using Statistical Product and Service Solutions (SPSS) 24.0 . Demographic information and the establishment status of health records were described using descriptive statistical analysis. The difference in the health record establishment of young migrants with various characteristics was analyzed by performing a chi-square test. Multivariate analysis was tested by binary logistic regression. The associated factors of the establishment of health records among young migrants in China were further investigated by performing binary logistic regression and using the odd ratio (OR) and 95% confidence intervals (CIs). A two-side inearity .Since the establishment of health records is classified as either “yes” or “no,” a binary logistic model was used to fit the data and explore the associated factors of the establishment of health records among young Chinese migrants. The formula of binary logistic regression is as follows:p denotes the probability of establishing health records, α is a constant term, and the fitting regression coefficients are expressed as βj, the potential explanatory variables are expressed as Xj. The OR indicator is able to be directly calculated in logistic regression by OR = eβ.where 50.8% were females among 115,730 young migrants who were aged 32.00 on average, with a standard deviation of 6.50. Most participants were rural (82.8%) and married (79.8%) with a middle school diploma (43.3%). Most young migrants bought medical insurance (91.7%), among whom about 58.2% had an average monthly household income of above 5001 yuan. As for migration characteristics, more young internal migrants (41.7%) were in eastern regions, and most of the participants were inter-provincial young migrants (47.8%), with the duration of migration 2–4 years (33.3%). The sample population of this study immigrated for working or engaging in trade (84.2%), with settlement willingness (84.2%). With regard to health status and PHS characteristics, the majority of young migrants had good self-rated health (87.0%), about 2.0% had hypertension or type 2 diabetes, the distance of medical facilities was ≤ 15 min (84.2%), 61.8% had heard about national BPHSs, and most received health education (75.1%). Regarding social integration, 49.0% of young migrants participated in social activities, 45.2% participated in public affairs activities, 93.7% were willing to integrate and type of people in touch was natives in the inflow area (34.5%).The results indicated that about 30.2% of young migrants established their health records in their local communities. Establishment rate of health records among young migrants in the east, central, west, and northeast areas were 26.0%, 41.3%, 30.9%, and 24.7%, respectively.Univariate analysis indicated that gender, age, marital status, Hukou status, education level, medical insurance, and average monthly household income were significantly associated with health record establishment Table .Table 1Univariate analysis indicated that sample area, extent, and duration of migration, reasons for migration and settlement willingness were significantly related to health record establishment Table .Table 2Univariate analysis indicated that self-rated health, distance of medical facilities, health education, and hearing about national BPHSs were significantly associated with health record establishment Table .Table 3Univariate analysis indicated that participation in social activities and public affairs activities, willingness to integrate and type of people in touch were significantly related to health record establishment Table .Table 4A co-linearity analysis showed a VIF < 10 and no co-linearity among the independent variables Table . Table 6The designated policy pointed out that health records should keep an establishment rate of above 75% . PreviouIn line with previous research, young migrants with a non-agricultural Hukou had a high possibility of establishing health records, which may be because migrants with an agricultural Hukou are more marginalized in many ways . Basic mMigration characteristics substantially contributed to the health inequality among young migrants who tend to be vulnerable to poor health conditions and utilize insufficient BPHSs due to external environmental factors and internal cultural shock in the cities where they live. It was noticed that northeast and inter-province migrant status, duration of migration ≤ 1 year, working or engaging in the trade as the reason for migration and no settlement willingness had a negative impact on the health record establishment of young migrants, which was parallel with previous studies , 29. It Young migrants who received health education and heard about national BPHSs showed a higher likelihood of establishing health records. These results indicated that health education and hearing about national BPHSs had positive impact on the health record establishment of young migrants. Apart from helping to improve the health conditions of young migrants indirectly by identifying their health risk factors, keeping healthy habits and shaping individual behavior, health education assists young migrants in gaining a better understanding of the advantages of establishing health records, removes privacy concerns and social distrust, and advances the health literacy and health consciousness of young migrants gradually. However, only less than 50% of migrants with health education or who know about national BPHSs established health records. These results suggested that providing health education or providing information on national BPHSs was not enough to establish health record in young Chinese migrants. Therefore, it is essential to adopt effective methods to implement health education and promotion in the community , 31. SpePrevious studies reported that community activities were conducive to improving the accessibility of health services among internal migrants . The resThe limitations of this study should be considered. First, cross-sectional data were used, preventing the interpretation of causal relationships. Second, subjective biases cannot be ruled out because of self-reported information on the establishment of health records and status. Thirdly, some variables considered in our study cannot be modified. There are no effective interventions to establish health records based on these variables. More modifiable factors should be further studied in future. Finally, the factors which were associated with the adoption of health records should be identified from the perspectives of health service providers and users. This study was based on the associated factors of health service users. More essential potential factors of health service providers should be further studied and confirmed in follow-up studies.This study showed that the health records of young migrants had a relatively low establishment rate. Sociodemographic and migration characteristics, health status, public health services, and social integration factors were all associated with health record establishment. Due to heterogeneity, policies and interventions should take into account different subgroups, especially vulnerable ones, strengthen the impact of health education on the popularization of health knowledge and the establishment of health records, and improve the health level of young migrants. The multifaceted association suggested that health administrations, communities, and young migrants need to make joint efforts of varying degrees to strengthen the equalization of BPHSs, and form external recourse supply and internal active participation to promote the establishment rate of health records.


Rangifer tarandus tarandus, n = 21) were scheduled twice for chemical immobilization with medetomidine–ketamine as part of a scientific experiment in June 2014. During the first round of immobilizations, seven animals developed severe respiratory depression (RD). Three individuals died, and 4 recovered. The ambient temperature during the 2 days of immobilization (June 3 and 4) was high (mean 13.9–17.6°C) compared to the normal mean temperature for these 2 days (7–8°C) based on statistical records. During the second round of immobilizations, using the same anesthetic protocol for the remaining animals as in the first round but conducted under cooler conditions (mean 6.6°C for the period June 9–18), no signs of RD were observed. Clinical and pathological investigations indicated that the animals suffered from circulatory changes possibly caused by high ambient temperatures and granulomatous interstitial pneumonia due to Elaphostrongylus rangiferi larvae. These conditions, together with the cardiovascular effects of medetomidine, were likely causes of RD and the fatal outcome. We conclude that chemical immobilization of reindeer with medetomidine–ketamine should be avoided in May–June due to the potential risk when animals partly in winter coats encounter rising ambient temperatures and usually have parasites developing in their airways.Semidomesticated Eurasian tundra reindeer ( Rangifer tarandus ssp.) and has been the recommended anesthetic drug combination for Eurasian tundra reindeer (Rangifer tarandus tarandus) for more than 20 years , 5 non-pregnant females and 16 males, were selected for an experimental study and brought from a mountain pasture to a corral .2, respiratory rate, and rectal temperature for the two rounds of immobilization are presented in The remaining 14 animals showed no signs of RD [respiratory depression minus (RD–)] during immobilization. Recordings of body mass, drug dose, time points for TBD and time head down (THD), heart rate, SpOHyperthermia, defined as body temperature ≥2°C higher than normal . The recpost mortem in animal R12. The heart contained no blood in neither atria nor ventricles. Various amounts of bloodstained subcutaneous edema were present in the thoracic inlet (R12 and R14), lower ventral neck (R12), upper part of the right (R14) or left (R11) shoulder, and in both flanks (R14). There was hyperemia in cutaneous vessels, most pronounced in animal R11. Scattered areas of subcutaneous petechiae were seen in R11 and R12. Histological examination of lung tissue revealed a multifocal, partly coalescing granulomatous interstitial pneumonia in all animals, with frequent occurrence of nematode eggs and developing stages of larvae, morphologically corresponding to Elaphostrongylus rangiferi   . The affn = 4) and RD– group (n = 14), chemically immobilized a second time prior to euthanasia 5–13 days after the initial immobilization, were not subjected to full necropsy. An inspection of body condition and organs was performed, gross findings were subjected to histological examination, and the presence of macroparasites was registered. Diffusely firm and “rubbery lungs”  were observed in R1 (RDR group) and in R2 and R18 (RD– group). In the lungs of R8 (RDR group), firm areas, partly as nodules of 0.5–1.5 cm in diameter, were distributed in cranial and caudal lung lobes on both sides. Histological examination revealed an acute and subacute purulent pneumonia with multifocal necrosis. Bacterial cultivation from lesions showed a rich growth of mixed microbiota, from which Streptococcus suis II was isolated.Animals of the RDR group (Cephenemyia trompe) were detected in two animals from the RDF group and two from the RD– group. Heavy infection with throat bot larvae was found in animal R1 (RDR group), with 107 larvae in the nasopharynx and nasal cavity. Trachea and the main bronchi were opened and inspected in all animals. The reindeer lungworm (Dictyocaulus eckerti) was detected in moderate numbers (10–20 worms) in R2 and R20 (RD– group) but not associated with gross pathological changes.A low number (1–7) of throat bot larvae  were on average 65% of the recommended dose for medetomidine–ketamine . Throat bot larvae may thus have influenced blood oxygenation but can hardly explain the RD for other animals hosting only a few larvae.Mature throat bot larvae that are ejected through the mouth or nostrils of reindeer (late April to June and July) by coughing  may causMore importantly, the necropsy findings in the RDF animals suggested circulatory collapse and pulmonary edema. The lungs contained little blood. There was no blood in the heart in any of the 3 RDF animals, which may be due to the conducted cardiac compressions. The spleens in animals R12 and R14 (RDF group) were moderately congested, but otherwise the abdominal organs contained sparse amounts of blood. Thus, the bulk of blood was distributed peripherally, in skin and subcutaneous tissues. This may be a cardiovascular response to high ambient temperatures in order to increase heat loss . In animThe pulmonary edema that developed in all RDF animals may also have been conditioned by the parasitic interstitial pneumonia, as lung inflammation predisposes to edema formation . WhetherE. rangiferi larvae. The adult nematodes live in muscle fasciae and deposit eggs in veins. The eggs are carried to the lungs where they develop to first-stage larvae that penetrate the alveolar wall and enter the air passages, to be carried by mucus up bronchi and trachea, swallowed, and leaving their host through the feces. E. rangiferi is prevalent in reindeer in Norway  during the first round of anesthesia. This highlights that any lung condition alone not necessarily causes severe RD during chemical immobilization.Animal R8 (RDR group) had a multifocal purulent bacterial pneumonia when examined 2 70%) indicated severe hypoxemia. Also, the RDR animals showed marked or severe hypoxemia during both rounds of immobilization , as compared to the RD– animals , displaying marked but not severe hypoxemia. The use of oxygen supplementation, which is strongly recommended and has been established as a standard procedure  is >95%. Hypoxemia can be defined as mild (90%−95%), marked (75%−90%), and severe (<75%) , 11. ForSince these animals were immobilized at a resting state, they were not suffering from an increased oxygen demand associated with increased metabolic rate and body temperature, which often is the result of stress and chasing animals prior to darting . FurtherIt may be controversial to claim that high ambient temperature was an underlying cause of mortality during the immobilization, since there was no registered increase in body temperature. However, the repeated immobilization of the same animals under cooler conditions, using the same drugs and doses for each individual, and without experiencing the same physiological challenges, leads to this suggestion. This further indicates that it is not only the ambient temperature at the actual time of the chemical immobilization that is important, but rather the hours and maybe days prior to the immobilization. Still partly in winter coat and experiencing a sudden high ambient temperature for the season, these animals were suffering from circulatory changes, with blood shunted to the peripheral capillary beds to contribute to loss of excess heat. This already challenged physiological equilibrium may have been further challenged by the adverse cardiovascular effects of medetomidine, causing bradycardia and decrease in cardiac output , 14.E. rangiferi larvae and, for 1 animal, airway obstruction from throat bot larvae, may have contributed to the morbidity and mortality during chemical immobilization. During the second round of immobilization, the RDR group still had higher heart rate and lower SpO2 than those of the RD– group , P.O. Box 383, 2381 Brumunddal.MT designed the study. MT, JS, NM, and TM conducted the immobilization. TJ conducted the necropsy and laboratory investigations. MT and TJ wrote the first draft of the manuscript. All authors contributed to the evaluation of the results and to the finalizing of the manuscript and accepted the submitted version.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.


SEPT9, TMEFF2, SDC2, NDRG4, VIM and ALX4). Optimal marker panels were determined by two statistical approaches. Methylation levels of SEPT9 were significantly increased in urine supernatant of CRC patients compared to controls (p < 0.0001). Methylation analysis in unfractioned urine appeared inaccurate. Following multivariate logistic regression and classification and regression tree analysis, a marker panel consisting of SEPT9 and SDC2 was able to detect up to 70% of CRC cases in urine supernatant at 86% specificity. First evidence is provided for CRC detection in urine by SEPT9 methylation analysis, which combined with SDC2 allows for an optimal differentiation between CRC patients and controls. Urine therefore provides a promising liquid biopsy for non-invasive CRC detection.Colorectal cancer (CRC) is the second leading cause for cancer-related death globally. Clinically, there is an urgent need for non-invasive CRC detection. This study assessed the feasibility of CRC detection by analysis of tumor-derived methylated DNA fragments in urine. Urine samples, including both unfractioned and supernatant urine fractions, of 92 CRC patients and 63 healthy volunteers were analyzed for DNA methylation levels of 6 CRC-associated markers ( Currently, there is a high clinical need for a non-invasive CRC biomarker test. Ideally the biomarker should have high accuracy and be able to provide means for detection of CRC, prognostication and disease monitoring during and after treatment3. Due to the absence of symptoms during early stages of disease, nearly half of patients are diagnosed at an advanced stage. Cancer stage at diagnosis however strongly correlates with survival, illustrated by the 5-year survival rate of 95% for stage I as compared to only 11% for stage IV2. Timely detection of CRC could improve mortality rates due to better chances of diagnosing CRC at a more curable disease stage4. Screening for CRC is therefore recommended in the western world as it is helps to lower the death rate due to CRC5. Screening tests are stool-based or visual. However, both are cumbersome and colonoscopy carries risks as it is invasive7. The ongoing development and increasing palette of CRC treatments require better tools to prognosticate patients. As molecular subtyping is key, colonoscopies and invasive biopsies are often warranted to decide on the appropriate treatment strategy9. Furthermore, more accurate disease monitoring and detection of minimal residual disease following treatment is necessary. Existing tools, including imaging and engrained protein-based biomarker CEA, lack sensitivity or come with risks. Non-invasive means for accurate detection of a molecular signature of CRC would mean opportunities for each of the early detection, prognostication and monitoring modalities. This urgent need has led to extensive research on molecular CRC biomarkers in recent years, with growing interest in liquid biopsies.Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the second leading cause for cancer-related death, accounting for 10–12% of all cancer cases10. Blood containing cell free DNA (cfDNA) is the most studied liquid biopsy to date. Cell free DNA consists of extracellular nucleic acids released into the circulation by means of necrosis, apoptosis and active secretion11. A small fraction of the total cfDNA consists of tumor derived DNA, so called circulating tumor DNA (ctDNA). Detection of ctDNA can be facilitated by analysis of DNA methylation, which is the enzymatically induced covalent binding of a methyl group (-CH3) to cytosine-guanine dinucleotides (CpG)12. It is a common epigenetic regulator of gene expression, frequently altered in cancer13. Increased methylation in CpG dense regions, called CpG islands and located in promoter regions of tumor suppressor genes, can lead to inactivation of tumor suppressor genes. As this is believed to be an early and critical event in CRC development, analysis of ctDNA methylation has potential to serve as a biomarker for CRC16.A ‘liquid biopsy’ enables the analysis of cancer derived molecules in biofluids, including peripheral blood, saliva and urine19. Despite advances in ctDNA diagnostics in blood, the process of venipuncture remains invasive and many challenges still have to be tackled. Analysis of ctDNA in urine features a non-invasive and logistically attractive way of testing.Circulating tumor DNA is excreted in urine through glomerular filtration, which propagates urine analysis as a true non-invasive method of cancer detection21. The low-MW group consists of smaller DNA fragments between 150 and 250 bp. Presumably, the low-MW fraction of urinary DNA is partially derived from the blood circulation, allowing detection of ctDNA in urine samples22. One method by which urine samples can be enriched for low-MW DNA is by centrifugation, which partly separates potential tumor DNA from non-specific high-MW DNA18. In contrast to, for example bladder cancer, in which high-MW DNA present in urinary sediment is suitable for tumor DNA detection23, the supernatant fraction is expected to be of most interest for detection of non-urogenital tumors, such as colon cancer, in urine.Urinary cfDNA can roughly be divided into two groups based on fragment size, comprising high- and low molecular weight (MW) groups. The high-MW group consists of heterogeneous DNA fragments of 1 kilobasepair (kbp) and larger, typically originating from the cell debris of the urogenital tractIn this study, we aimed to evaluate the diagnostic potential of urine DNA methylation analysis for detection of CRC.24. Healthy volunteers, serving as controls, were selected for eligibility through a pre-defined selection process. By taking a questionnaire, it was verified if control subjects were not diagnosed with cancer at any point during their life, and matched the age range of the CRC patient test groups. Urine samples from controls were also collected in containers containing 40 mM EDTA and processed upon arrival. For both CRC patients and controls an independent set of consecutively collected urine samples was used for our studies on unfractioned urine and urine supernatant. To obtain the urine supernatant fraction and enrich for low-MW, DNA samples were centrifuged at 3000 g for 15 min. Unfractioned and supernatant urine specimens were frozen at − 20 °C until further use.Consecutive patients suffering from CRC who visited the Surgery Department Amsterdam UMC, a tertiary referral center in, Amsterdam, The Netherlands between January 2018 and February 2019, were included in the study. All patients were older than 18 years, were diagnosed with a pathology proven CRC, and underwent no recent anticancer treatment during the last year. Patients with other malignancies in the previous 3 years were excluded. All participating patients provided urine samples during visits to the outpatient clinic prior to surgery. Samples were collected in 40 ml containers containing 40 mM Ethylenediaminetetraacetic acid (EDTA) and subsequently processed within 6 h and stored at 4 °C. Both addition of EDTA and storage at 4 °C preserves urine DNA for accurate methylation analysisSample collection and study design were approved by the Medical Ethical Committee board of the Amsterdam UMC for both CRC patients and healthy volunteers (no. 2018.035 and no. 2018.657). Written informed consent was obtained from all participants of this study. All experiments were performed in accordance with relevant guidelines and regulations.For DNA isolation from unfractioned urine and urine supernatant the Quick DNA urine kit  was used. This isolation method proved superior for the isolation of small DNA fragments (as low as 50 bp) over other DNA isolation methods (data not shown). DNA from the CRC cell line RKO (American Type Culture Collection) was isolated using the PureLink genomic DNA kit .Isolated DNA was eluted in 50ul elution buffer. DNA concentrations were measured using the Qubit™ dsDNA HS Assay . For methylation analysis, up to 400 ng of isolated DNA was treated with bisulfite using the EZ DNA Methylation kit . All procedures were performed according to manufacturer’s guidelines.SEPT9, TMEFF2, SDC2 and NDRG4, VIM and ALX4) were selected from a systematic literature search based on accuracy3.Six CRC- associated DNA methylation markers  and reference gene (β-actin: ACTB) were designed based on sequences as described previously29. By adjusting amplicon sizes to a maximum of 80 bp, detection of CRC-derived low-MW DNA was facilitated. Multiplex development was executed according to optimization parameters as described by Snellenberg et al.30. In brief, marker specificity was individually evaluated by MSP with unmodified and modified DNA isolated from CRC cell line RKO. Sensitivity analysis demonstrated that both qMSP multiplexes were able to detect methylated RKO DNA diluted in water up to dilutions of 0.1% and 0.5%, respectively. Primer and probe limiting assays were performed to determine their ideal concentrations in both singleplex and multiplex qMSP’s. qMSP analysis was performed on a ViiA7 real-time PCR-system , using Epitect Multiplex PCR Mastermix  Methylation marker abundance was calculated relative to ACTB levels (Ct-ratio), using the following formula: 2 − (CtMARKER − CtACTB) * 100. Further details are provided in supplementary S1.Two multiplex quantitative Methylation Specific PCRs (qMSPs), each consisting of 3 targets , results of stage IV patients were split up between peritoneal metastasis solely and stage IV including all types of (including hematogenous) metastasis. In this study these were only liver metastases.Analyses of relationships between methylation and clinical parameters were only performed for marker 31 for further details on CART method. For the purpose of prediction, the predicted class was obtained by leave-one-out cross validation. For both building the decision tree as well as performing prediction, the R package Recursive Partitioning or rpart was used.To determine the ability of a combination of markers to differentiate between controls and patients, two approaches were explored for determining both the best marker panel and marker thresholds for a maximal test accuracy. In the first method, multivariate logistics regression (MLR) was used to model the probability of a urine sample being from a CRC patient, with all six methylation markers as independent variables. First, we fit a model with the six main effects only, and selected markers by stepwise selection. Then, to investigate whether the in-model effect of an individual marker was affected by other markers, we added the two-way interaction terms that include the selected main effects, again followed by stepwise selection. A leave-one-out cross-validation was then used to evaluate the performance of the model for prediction. Next, the predicted probability from this cross-validation was used for sample classification, according to a maximal Youden’s index, i.e. the sum of sensitivity and specificity minus 1. For fitting the MLR model, the R function Generalized Linear Models or glm was used. Apart from the MLR, we applied an algorithm-based method called classification and regression tree (CART) for binary classification of cases and controls on the same set of methylation markers. For this alternative analysis, a decision tree was obtained allowing for classification of urine samples based on marker values. We refer toThe performance of both methods was determined from obtained sensitivities and specificities. For logistics regression, the Receiver Operator Characteristic (ROC) curve was plotted together with maximized Youden’s index.Statistical analyses were performed using SPSS software  and R . Data visualization and construction of graphs was facilitated by GraphPad . Additional details of all statistical analyses can be found in supplementary file S1.In total 47 CRC patients and 20 healthy controls were included in the unfractioned group, and 45 CRC patients and 43 controls in the supernatant group. Clinical characteristics of CRC patients and controls with valid qMSP results are depicted in Table The DNA yield of both unfractioned urine and urine supernatant collected from CRC patients and controls were evaluated to assess the utility for methylation analysis. The sample DNA concentrations are shown in Table SEPT9, TMEFF2, SDC2, NDRG4, VIM and ALX4 in unfractioned urine samples of CRC patients (n = 47) and controls (n = 20) was investigated to evaluate their potential for CRC detection. Elevated methylation levels were detected in a subset of CRC patients for SEPT9, and at low frequencies for VIM and ALX4 . The remaining markers were detectable in 2–36% of CRC patients and 0–20% of controls.DNA methylation of LX4 Fig. a. FollowLX4 Fig. b. Methyl18, would allow for a better discrimination between patients and controls. The same methylation markers were tested on urine supernatants from an independent cohort of CRC patients (n = 45) and controls (n = 44). As shown in Fig. SEPT9 methylation levels were significantly elevated in CRC patients compared to controls (p < 0.0001). No significant differences were found for the remaining five markers. Assessment of the absolute detections rates also demonstrated that SEPT9 methylation analysis detected significantly more CRC patients than controls (p < 0.01). Patients with solely peritoneal metastases had a trend towards lower levels of urine ctDNA, as compared to patients with liver metastases.In the group of CRC patients, no difference in ase Fig. . HoweverThe diagnostic potential of a combined panel of DNA methylation markers was evaluated to differentiate between healthy controls and CRC patients. Both a multivariate logistics regression (MLR) and classification and regression tree (CART) analysis methods were used to assess accuracy. Furthermore, discrepancies were determined between models with regard to sample classification. To allow for a complete analysis, CRC samples (n = 2) and control samples (n = 1) that had an invalid ACTB in one of the two multiplexes, were discarded in this process which resulted in a total of 43 CRC and 42 control urine supernatants to be evaluated.SEPT9 was significantly associated with the probability of being in the case group (p < 0.0001). Therefore, a logistics regression was fitted, with SEPT9 and the interaction terms with the other five markers. The stepwise selection procedures selected SEPT9 methylation and the interaction term between SEPT9 and SDC2 methylation to be strongly associated with the probability of being a CRC patient. Figure SEPT9 and SDC2. In this model, when methylation levels of SEPT9 were low, the probability of being a case was small, irrespective of SDC2 levels. In the higher values of SEPT9 however, we noticed that gradual increases of predicted probabilities of being a case were affected by the values of SDC2. This explains the interactive effect of SEPT9 and SDC2.When fitting the MLR model with main effects, only SEPT9 is the most important predictor for the classification, but again SDC2 constitutes an interaction variable. When SEPT9 methylation was higher or equal to -0.098, subjects were classified into cases . Finally, when the value of SEPT9 was lower than -2.9, the threshold of SDC2 = − 1.8 determined the controls  and cases .In the second approach, we used a CART model to classify cases and controls based on the values of each markers. The resulting decision tree is depicted in Fig. ses Fig. , node 3.Finally, leave-one-out cross validation was utilized to evaluate the prediction performance of MLR and CART. Using the MLR model, we obtained the estimated probability of being a case while the CART decision tree assigns a sample as case or control Figure . Hence, SEPT9, as compared to healthy control patients. SEPT9 methylation, combined with marker SDC2, offers a potential novel tool for detection and monitoring of CRC. Using short-amplicon methylation specific PCRs, we have successfully detected CRC-associated DNA methylation in urine supernatant. Out of six markers tested, SEPT9 showed best accuracy to serve as a potential urinary biomarker for CRC detection. By combining SEPT9 and SDC2, up to 70% of CRC cases could be detected at a specificity of 86%.This study demonstrates for the first time that urine of CRC patients contains elevated levels of the DNA methylation marker 24. This allows for reliable testing of samples collected in an ambulant setting. Additionally, there are no limits to available quantities. For screening programs of other types of cancer, urine is currently evaluated as a non-invasive alternative to physician-involved diagnostics35. The results from the present study suggest that urine has the same potential for CRC detection, by showing ctDNA is detectable through means of DNA methylation analysis.Despite extensive research, a need still exists for a non-invasive biomarker to detect CRC during clinical management. While many studies have been performed on the use of ctDNA in plasma for these purposes, a possible role for urine has not yet been well elucidated. Urine as a biofluid poses several advantages over blood. It does not require trained professionals to acquire, it lends itself for easy repeated sampling and it has been shown that urine poses a very stable medium for DNAKRAS DNA mutations22. Some methylation markers tested in the present study have been described before for CRC detection in urine samples. Methylation marker VIM was assessed in two separate studies36. In a study that selected low molecular weight DNA from urine samples using carboxylated magnetic beads, 12 of 17 LMW (71%) urine DNA samples of CRC patients were found to be positive for VIM methylation, compared to two out of 20 (10%) control samples28. Another earlier publication however, showed a poor performance of VIM methylation detection in urine (i.e. 8% sensitivity at 100% specificity). This study also assessed the methylation markers WIF-1 and ALX4 in urine, for which respectively a sensitivity of 27% and 15% at 99% and 100% specificity was found36. Detection of NDRG4 methylation in urine has been described earlier by Xiao et al. with 55 of 76 (73%) CRC cases testing positive for urine methylation, at a specificity of 85% based on 36 controls29.This report is among the first few publications exploring the feasibility of molecular analysis in urine for the purpose of non-invasive CRC detection. In a pioneer study by Su et al., the distinction between high and low-molecular weight (HMW and LMW) urine DNA for CRC detection was made, showing the latter provides higher accuracy for detection of CRC-specific NDRG4 and VIM. In the present study, centrifugation appeared effective for enrichment of highly fragmented tumor DNA (low-MW), as supernatant samples enabled a more adequate differentiation between disease and healthy controls compared to unfractioned urine that contains both high-MW and low-MW DNA. Furthermore, we used a dedicated urine DNA isolation kit. Interestingly, all mentioned previous studies did not fractionate urine samples prior to DNA isolation and did not use specialized urine DNA kits. Song et al. stored urine samples on − 70 °C directly following collection, and isolated for low-MW DNA using magnetic beads-based selection and resin-based DNA isolation after defrosting of the urine samples28. Xiao et al. isolated DNA directly following collection, however no pre-PCR DNA size selection was performed and no information was given on the isolation method29. Amiot et al. did not provide any details with regard to urine processing and used the same generic DNA isolation kit for urine, plasma and stool36. Concerning differences in sample population, Song et al. collected control samples from patients that underwent a colonoscopy yielding negative results. The other studies did not provide further information on their controls. Furthermore, differences in test group ethnicity might have influenced baseline methylation levels37. Another important reason of discrepancies in marker performance between present and earlier studies, could be differences in the actual targeted marker CpG dinucleotides undergoing PCR amplification.Differences in methodology or sample population may explain the discrepancies in CRC detection rates of methylation markers KRAS mutations in urine, only Fuji et al. explored the modality of advanced CRC treatment monitoring, showing a decline of urinal/urinary KRAS mutations during effective systemic therapy38.Besides limited data on the use of urine for detection of CRC, little evidence is currently available for prognostication and disease monitoring. By detection of SEPT9 methylation in urine. Urine SEPT9 methylation analysis showed a sensitivity for CRC detection coming close to those reported for SEPT9 methylation analysis in plasma samples, which vary from 75 to 81% (at ≥ 96% specificity)39. SEPT9 methylation analysis for CRC detection in plasma is now available as an FDA-approved commercial test . Established CRC plasma methylation markers tested in this study, other than SEPT9, showed lower detection rates. Possible explanations include both biological and technical causes. Urine as a biofluid might have properties that lead to decreased detection of certain ctDNA fragments. Pores present in the glomerular basal membrane (GBM) may select not only on molecular weight, but also its net negative electric charge could play a role in preventing blood-urine translocation of the negatively charged DNA40. Other thermodynamic properties of DNA that lead to its polymorphic potential, as well as complex formation with for instance proteins, might also influence the probability of glomerular translocation. As these properties are hugely influenced by particular nucleotide sequences of ctDNA, certain methylation markers might have a decreased performance in urine. A comparison between blood and urine samples from the same test subjects would therefore be very interesting to estimate the rate by which the methylation signal gets lost due to GBM filtration.To our knowledge, this is the first study describing detection of SEPT9 methylation levels and clinical stages of CRC of patients. Although the detection of ctDNA appears more likely when sampling occurs during higher clinical stages of neoplastic disease, this was not the case in the present study. Similarly, the study on VIM methylation in CRC urine samples did also find no correlation with CRC stages28. For stage IV patients however, we found that the presence of the primary tumor may be an attributive factor for detecting CRC-associated methylation in urine DNA. This probably also relates to the fact that the majority of included stage IV patients without the primary tumor present were suffering from peritoneal metastases. Data of our group shows that patients with peritoneal metastases have a smaller tendency to have detectable ctDNA41. Furthermore, the majority of peritoneal metastases are classified as CMS4 (Consensus Molecular Subtypes). In this tumor subtype, methylation levels are often very low43.No differences were found between urine A novel approach in this study is the use of two different methods of statistical analysis to determine the complementarity between methylation markers to achieve maximal accuracy for CRC detection. The MLR and CART models agreed on classification of most urine samples, supporting the validity of our results. The MLR model gave a slightly higher sensitivity (70%) compared to the decision tree (67%), whereas the CART model yielded a slightly higher specificity . In this study, the MLR model was optimized to achieve a maximal Youden’s index but could, depending on the clinical context, be adjusted to achieve a higher sensitivity or specificity. Furthermore, it should be noted that especially the CART model provides practical means to evaluate an individual’s test results, by simply noticing the marker value and subsequently following the branches of the tree. The MLR model on the other hand provides a probability of being a case based on methylation marker values. Therefore, this model is more generalizable to the whole population compared to the decision tree. Hence, both methods can be used depending on the requirements that follow the specific clinical demand.3. Genome wide methylation analysis of CRC urine specimens could possibly discover novel markers with better performance in urinary cfDNA. Furthermore, in this study, 40 ml of urine was used for DNA isolation, being a technical limitation of methodology. Other studies studying urine for cancer detection have utilized larger volumes (up to 120 ml) of urine44. Methylation analysis with higher inputs of urine for DNA isolation might increase test accuracy and reduce test failures.A limitation of this feasibility study includes the relatively small set of samples. Also, the control group of the urine supernatant cohort included somewhat younger subjects and relatively more females than in the patient group Table . Althoug45. Recent large-scale efforts to screen for cancer in a population setting, have shown DNA methylation to be particularly suited for cancer detection and tissue of origin localization46. Nevertheless to increase test accuracy, it seems attractive to combine methylation analysis with additional molecular ctDNA markers, such as DNA mutations and/or copy number variations47 or with the analysis of CRC-associated metabolites48.The promising results of the present study warrant further verification and validation studies, comprising larger samples series and application of pre-defined thresholds of the models used in this study. Depending on the designated clinical setting however, a validation study might require thousands of test subjectsSEPT9 and SDC2, CRC could be detected with high accuracy. This is the first step in conceiving a urine-based test for CRC and ultimately, other cancers as well.In conclusion, this study demonstrates the feasibility of urine supernatant for detection of CRC. Through means of DNA methylation analysis of a marker panel consisting of Supplementary Information.


Loxodonta africana). As a result, not much is known about the ability of elephants to thrive in such a unique environment. External factors such as a nutritionally poor diet can be perceived as a stressor by mammals, and establishing links between these two factors was the focus of this study. The results of monitoring individual stress-related hormone levels and body conditions suggest that elephants can obtain adequate nutrition from Fynbos vegetation.The Western Cape Province of South Africa is characterized by Fynbos vegetation. This unique endemic vegetation type belongs to the Cape Floral Kingdom, the smallest of the six floral kingdoms in the world, and only a few provincial, national and private game reserves in this area currently support populations of African elephants (Loxodonta africana), and thus, little information exists regarding the suitability of the nutritionally poor Fynbos vegetation for these megaherbivores. Using already established non-invasive methods, the monitoring of individual body conditions and fecal glucocorticoid metabolite (fGCM) concentrations, as a measure of physiological stress, was performed to examine a herd of 13 elephants in a Western Cape Province Private Game Reserve, during two monitoring periods (April and June 2018), following a severe drought. The results indicate that overall median body condition scores  and fGCM concentrations (April: 0.46 µg/g dry weight (DW), range 0.35–0.66 µg/g DW; June: 0.61 µg/g DW, range 0.22–1.06 µg/g DW) were comparable to those of other elephant populations previously studied utilizing the same techniques. These findings indicate that the individuals obtain sufficient nutrients from the surrounding Fynbos vegetation during the months monitored. However, a frequent assessment of body conditions and stress-associated fGCM concentrations in these animals would assist conservation management authorities and animal welfare practitioners in determining ways to manage this species in environments with comparably poorer nutritional vegetation.Fynbos is a unique endemic vegetation type belonging to the Cape Floral Kingdom in the Western Cape Province of South Africa, representing the smallest of the six floral kingdoms in the world. Nowadays, only a few game reserves in this region support populations of African elephants ( Loxodonta africana) [2 area which is recognized as an important global hotspot of endemic biodiversity. The CFR is characterized by a number of different biomes, with the Fynbos Biome being the most prominent [2 of the total area and includes species such as evergreen sclerophyllous shrubs, which rely on fire for regeneration and, due to the soil-poor nutrient contents, dominate the landscape, replacing trees [Previous studies reported the presence of considerable numbers of large herbivores >20 kg) being resident as recently as 350 years ago in the Fynbos biome in the Western Cape Province of South Africa, including an estimated population of about 10,000 African bush elephants , commonly used when managing domestic livestock and recognized as a standardized veterinary procedure ,33,34. TThe aim of this study was to examine a herd of 13 free-roaming elephants within a reserve in the CFR characterized by Fynbos vegetation across a season following severe drought through (i) individual BC scores, using the methodology of Morfeld and colleagues , and , African buffalo (Syncerus caffer), Southern giraffe (Giraffa giraffa) and white rhino (Ceratotherium simum), in addition to large herds of antelope. The grazers tend to congregate on the short lawn grasslands, while the elephants move around the reserve in search of suitable forage patches. The focal animal group consisted of 13 free-roaming elephants, introduced into the reserve between 2008 and 2014. The composition of the group included two adult bulls (34 and 29 years old), which generally roam together, and two matriarchal herds (α and β herd). The α herd is composed of two adult females (age unknown), four juveniles (3–7 years old) and one calf (1 year old), while the β herd is formed by only two adult females (both 26 years old) and two juveniles (5–6 years old). At the time of the study, all the elephants observed appeared to be in normal physical condition, and no sign of injury or any other form of ill health was recognized.The study was conducted in a fenced private game reserve of 11,000 hectares located in the Western Cape of South Africa , during the months of April and June 2018. Mean daily temperatures range between 11 and 21 °C and precipitation is generally a-seasonal, though rainfall occurs more frequently between autumn and spring (March–September). The long-term (1980–2019) average annual rainfall at the closest weather station at the Mossel Bay Lighthouse is 320 mm, and the total rainfall recorded in 2018 was 302 mm, with the majority occurring during the end of the wet season (September–October) . Water ig winter . The sitFor body condition scoring (BCS), a total of 78 suitable pictures , from the rear, the side and at a 45° angle from the body were taken. Two assessors evaluated the pictures independently and scored each individual for each month, using an established BCS index for African elephants . The meaFecal samples were collected opportunistically from as many individuals as possible during field work . Samples were collected within an hour of defecation , placed Frozen feces were lyophilized, and the resulting dry matter was pulverized and sifted through a mesh to remove undigested fecal matter . SubsequMatching individual body condition scores of elephants monitored in April and June 2018 were compared using the Wilcoxon Signed Rank test. The individual fGCM concentrations of elephants monitored in April and June 2018 were compared using the Mann–Whitney Rank Sum test. When repeated values were available from the same animal during a month, a median value was used for analysis. To compare matching individual fGCM concentrations of elephants monitored during both months (n = 7), a paired t-test was utilized. Data subsets were tested a priori for normality using the Shapiro–Wilk test and, if required, for equal variance using the Brown–Forsythe test. Individual BC scores and individual median fGCM concentrations were correlated for April and June separately using the Pearson Product Moment Correlation test. All tests were two-tailed, with the level of significance set at 0.05. Sigma Plot version 12.5.  was used for all statistical analyses.Individual BCS ranged between 2.0 and 3.0 for the 13 focal animals during the first (April 2018) and second (June 2018) observational periods and did not change significantly between the two periods . Out of the 13 elephants observed, a lower BCS was assigned to three animals in April compared to in June, whereas the opposite was given for only one animal . 10,9 = 116.5, P = 0.034) and also showed a wider range (April: 0.35–0.66 µg/g DW vs. June: 0.22–1.06 µg/g DW). When comparing matching values, six out of the seven animals showed higher median fGCM concentrations in June compared to in April, but the median fGCM concentrations did not differ significantly . Out of the 13 focal animals, fGCM concentrations could be determined for ten individuals for April and for nine individuals for June 2018, respectively. For seven animals, the fGCM concentrations could be obtained for both observational periods . The oveIndividual median fGCM concentrations and BC scores were not correlated during the monitoring period in April  or June 2018 .In this study, variations in individual BCS and fGCM concentrations were investigated in a herd of 13 elephants, free-roaming in nutritionally poor fynbos vegetation. The overall median individual body condition scores were 3.0 for both sampling months (April and June) and only ranged from 2.5 to 3.0 for the majority of the elephants throughout the study. Furthermore, the fGCM concentrations determined over the same study period were comparable to the values determined in previous studies on elephants roaming in other parts of Africa ,42,44,45Cynodon dactylon, which may provide important winter forage for elephants in the Eastern Cape [The fGCM concentrations determined in this study appear to be comparable to the baseline fGCM values determined in previous studies on elephants using the same non-invasive approach to determine stress-related hormone levels ,40,41,45ern Cape , remaineern Cape . The eleern Cape , for comern Cape , competiern Cape , or the ern Cape . Howeverern Cape ,60. ThusOryctolagus cuniculus), Steller sea lions (Eumetopias jubatus), and African and Asian elephants (Elephas maximus) [BC scores and fGCM concentrations were not correlated in our study animals. This is in line with the findings of Kumar et al. , who stumaximus) ,25,62,63Our study provides evidence that the monitored elephants living in the Fynbos biome maintain a BCS within acceptable limits and show stress-related fGCM concentrations comparable to the baseline fGCM values determined for other African elephant populations, ultimately suggesting that elephants can exist in reserves in CFR areas. However, in our opinion, reserve management would benefit from continuous monitoring over an extended period of time to truly evaluate the conservation and welfare implications.