In Africa, livestock production currently accounts for about 30% of the gross value of agricultural production. However, production is struggling to keep up with the demands of expanding human populations, the rise in urbanization and the associated shifts in diet habits. High costs of feed prevent the livestock sector from thriving and to meet the rising demand. Insects have been identified as potential alternatives to the conventionally used protein sources in livestock feed due to their rich nutrients content and the fact that they can be reared on organic side streams. Substrates derived from organic by-products are suitable for industrial large-scale production of insect meal. Thus, a holistic comparison of the nutritive value of Black Soldier Fly larvae (BSFL) reared on three different organic substrates, i.e. chicken manure (CM), brewers’ spent grain (SG) and kitchen waste (KW), was conducted. BSFL samples reared on every substrate were collected for chemical analysis after the feeding process. Five-hundred (500) neonatal BSFL were placed in 23 × 15 cm metallic trays on the respective substrates for a period of 3–4 weeks at 28 ± 2 °C and 65 ± 5% relative humidity. The larvae were harvested when the prepupal stage was reached using a 5 mm mesh size sieve. A sample of 200 grams prepupae was taken from each replicate and pooled for every substrate and then frozen at −20 °C for chemical analysis. Samples of BSFL and substrates were analyzed for dry matter (DM), crude protein (CP), ether extracts (EE), ash, acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids (AA), fatty acids (FA), vitamins, flavonoids, minerals and aflatoxins. The data were then subjected to analysis of variance (ANOVA) using general linear model procedure. BSFL differed in terms of nutrient composition depending on the organic substrates they were reared on. CP, EE, minerals, amino acids, ADF and NDF but not vitamins were affected by the different rearing substrates. BSFL fed on different substrates exhibited different accumulation patterns of minerals, with CM resulting in the largest turnover of minerals. Low concentrations of heavy metals (cadmium and lead) were detected in the BSFL, but no traces of aflatoxins were found. In conclusion, it is possible to take advantage of the readily available organic waste streams in Kenya to produce nutrient-rich BSFL-derived feed. In the recent past already major shifts in diets have happened, favoring more animal-based foods, in particular milk, meat, fish and eggs, and these preferences are expected to increase with time2. These changes in dietarian pattern have been accelerated by economic growth, coupled with rapid migration from rural to urban areas, as well an increasing awareness in nutritional needs. By the middle of the current century, cereal and meat production are expected to increase from 2.1 billion and 258 million tons produced per annum between 2005 and 2007 to 3.0 billion and455 million tons, respectively, raising worldwide concerns regarding the status of food security3.The global food demand is expected to increase by 70% by the year 2050 in order to meet the demands of the 9.7 billion people who are forecasted to inhibit the globe by that time5. Kenyan poultry farming is a significant source of income, especially in rural areas, and contributes to more than quarter of the agricultural Gross Domestic Product (GDP) and accounts for 8% of the total GDP in Kenya6. Yet feed costs make up more than 70% of the production costs9, highlighting the important role economic feeds and their availability could play in successful poultry farming. Due to food-feed competition, feed constituents that are suitable for direct human consumption such as soybean and fish are expensive and collectively increase the costs of feeds8. In addition, global catches from the marine fish stocks have dwindled over the years due to overexploitation10. This increases the price of fishmeal, which is not only used in feeding livestock but rather is a major source of protein in farmed fish feed12. Moreover, the intensification of soybean production, especially in the tropics, resulted in land grabbing and deforestation in addition to other negative social and environmental consequences13. For the reasons mentioned above, there is an urgent need to replace conventional feed ingredients such as soybean and fish with innovative, economically beneficial and environmentally sustainable ones14.In the developing world, the livestock sector can act as a gateway towards alleviating poverty and enhancing food security15. In addition, insects are precious reservoirs of proteins, fatty acids, micronutrients and contain high amounts of energy18. The latter show a good profile of amino acids in general, and of the most-limiting essential ones like lysine, threonine and methionine, often lacking in plant-based protein sources for non-ruminants19.Large-scale rearing of insects is a promising and innovative alternative as several insects’ species can feed of various types of organic waste streamsHermetia illucens L. (Diptera: Stratiomyidae), the common house fly Musca domestica L. (Diptera: Muscidae) and the yellow mealworm Tenebrio molitor L. (Coleoptera: Tenebrionidae) are among the insect species that have been recognized as promising alternative sources of protein for animal feed22. The first two naturally occur in animal droppings but also flourish on other organic waste substrates such as coffee bean pulp, vegetables residues, catering waste, municipal organic waste, straw, dried distillers grains with solubles (DDGS), and fish offal15, and can add value by reducing organic waste biomass by 50–60% and turning them into high protein biomass23. The yellow mealworm can be reared on vegetables and DDGS25. The dry weight of Black Soldier Fly larvae (BSFL) contain up to 50% crude protein(CP), up to 35% lipids and have an amino acid profile that is similar to that of fishmeal26. They are recognized and utilized as alternative sources of protein for feed of poultry, pigs, and several species of fish and shrimp27.The Black Soldier Fly (BSF) 28. It does not carry diseases, and actively feeding BSFL secrete an info-chemical that keeps away other species of flies, thereby repelling potential insect pests and disease vectors such as M. domestica29. The same authors also reported that BSFL significantly influence the reduction of Escherichia coli and Salmonella enterica presence in cow dung while Liu and colleagues30 reported the same influence on Escherichia coli in chicken manure. The economic feasibility of the use of insects as feed largely depends on cost effective and readily available organic waste streams, both in the developed and in particular in the developing world. So far, very few studies assessed the holistic nutritional contents of BSFL in terms of quality using experimental diets that were established within the means of the developing world. Unlike rationed diets, organic waste streams in the developing world are heterogeneous in nutritional composition and might be an environment where heavy metals can accumulate. Therefore, the current study sought to perform a comparative holistic analysis of the quality of the nutritional composition of BSFL reared on organic waste streams that are largely and readily available in urban areas of Kenya and the developing world in general. A comparative study that is essential when deciding which organic waste streams are potentially suitable for industrial large-scale BSFL production in Kenya.The adult fly can typically live for one to two weeks without the need to feed as it appears it can rely on fat body reserve that was acquired during larval stages and can even live longer when fed with water2 = 0.939; p = 0.0149) . Organic matter was lower in KW while the values obtained from CM and SG were comparable . Crude protein content was higher in KW in comparison to CM and SG . Moreover, the values of NDF  and ADF  contents in SG were higher in comparison to the two other rearing substrates. The EE contents  of the three substrates were rather low (2.7–7.2% DM) with CM the lowest.LC-Qtof-MS analysis for mycotoxins did not identify any traces of aflatoxin in the BSFL. There was an effect of substrate in all the proximate nutritional parameters Table . The DM 2 = 0.838; p < 0.0001) was high in the CM and SG fed larvae and lowest in the KW fed ones, though larvae fed on the latter substrate showed higher crude fat  content than the others. Organic matter  was lower in CM fed larvae as compared to larvae fed on the two other substrates. Both NDF  and ADF  contents were higher in SG fed BSF.There was an effect of substrate in all the proximate nutritional parameters of the BSFL except for ash Table . The DM 2 = 0.851; p = 0.0011) and KW . However, there was no high correlation between DM content of SG and that of the BSFL (p = 0.6391). Moreover, there was a high correlation between OM content of the rearing substrates and those of the BSFL for CM , KW  and SG . Moreover, there was a high correlation between the ash content of CM and SG and that of the BSFL   while there was no high correlation between the ash content KW and that of the BSFL (p = 0.4196). There was a high correlation between CP content of two of the three rearing substrates and those of the BSFL . Moreover, a high correlation was observed between the EE contents of substrates and those of the BSFL . There was a high correlation between the NDF content of the rearing substrates and those of the BSFL . Likewise, there was a high correlation between the ADF contents of the rearing substrates and those of the BSFL for CM , KW  and SG .There was a high correlation between DM content of the rearing substrates and those of the BSFL for CM , magnesium  and sodium  significantly differed among the tested BSFL whereas potassium and calcium did not.The three rearing substrates exhibited different accumulation patterns of minerals Table . Five ma2 = 0.641; p = 0.0005), calcium  and magnesium  significantly differed among the tested BSFL whereas sodium did not.BSFL fed on the different substrates exhibited different accumulation patterns of minerals Table . Five ma2 = 0.902618; p < 0.0001 for CM and DF = 1; F = 84.50; R2 = 0.913509; p < 0.0001 for SG). For calcium concentrations, there was a high correlation between the concentrations in two of the three rearing substrates tested i.e. CM and SG and those of the BSFL . Similarly, there was a high correlation between sodium concentrations in the three rearing substrates tested . Moreover, there was a high correlation between magnesium concentrations of the three rearing substrates tested and those of the BSFL .There was no significant correlation between phosphorus concentrations of the rearing substrates and those of the BSFL . Moreover, there was no significant correlation between potassium concertation of KW rearing substrate and that of the BSFL (p = 0.0153). However, there was a high correlation between potassium concentrations of CM and SG rearing substrates and those of the BSFL , hydro-proline  and tyrosine  compared to the larvae fed on the other tested substrates. Glutamine was detected in larvae reared on KW. Glutamic acid  was only detected in CM fed BSFL, while KW fed BSFL in most cases showed the highest concentrations of the different amino acids especially in the Phenylalanine  content, followed by SG and CM fed BSFL  and kaempferol  lower concentrations were recorded in KW fed BSFL as compared to the other substrates.A total of five flavonoids were detected, with two of them showing a significant substrate effect in their concentrations Table . For botThree vitamins were detected though the substrate had no significant effects on their concentrations, i.e. Gamma tocopherol p = 0.3767), Alpha tocopherol (p = 0.0713) and Provitamin D3 (p = 0.1399)  reported by Spranghers and colleagues37 for BSFL reared on various organic waste streams. Moreover, Nguyen and colleagues38 reported a CP value of 39% for BSFL reared on fruits and vegetables waste while Sheppard and colleagues23 reported 42% CP for BSFL reared on chicken manure. However, the previously mentioned CP values were obtained using the Kjeldahl standard nitrogen-to-protein conversion factor of 6.25 while the values we reported were obtained using the conversion factor of 4.76. The total nitrogen content in insects in general and BSF in specific contain nitrogen originating from both protein and non-protein sources such as chitin. Hence, separating non protein from protein nitrogen was necessary in order to obtain accurate crude protein content and to avoid over estimated values that were previously reported using the standard conversion factor39. When calculated using the standard nitrogen-to-protein conversion factor of 6.25, the range of CP values we obtained from this study (39 to 54%) surpassed the range of values previously reported in literature38. Our results were closer to that obtained by Caligiani and colleagues39 and Janssen and colleagues35 who both used a nitrogen-to-protein conversion factor of 4.76. Similarly, the EE content we found in BSFL was higher than those reported from full fat soybean meal36 and commercially available fishmeal40. Newton and colleagues41 and St-Hilaire and colleagues42 reported even higher EE values in BSFL than we found in our study. Previous studies reported an influence of the rearing substrate on the EE content. For instance, Nguyen and colleagues38 reported higher EE content for larvae reared on fish and liver in comparison to chicken feed. However, there was no apparent influence of the rearing substrates on the EE content of BSFL in our study.In this study, we measured the potential of recycling three readily available organic waste streams in Nairobi, Kenya, and arguably also in other megacities in the developing world, and their influence on the nutritional quality of BSFL as a proposed alternative protein source for livestock feed. As the quality of livestock feed is mainly measured in the concentrations of CP present in the feed’s dry matter, we conducted a proximate analysis applying the standard methods described by the Association of Official Analytical Chemist44. When referring to bone and egg formation in layer hens, calcium in particular plays a crucial role as it contributes to more than 90% of the mineral matrix in the bones45. Therefore, deficiencies in calcium and phosphorus can result in bone loss, growth retardation and abnormal posture45. The range of calcium concentrations reported in this study (1.7 to 3.2%) was lower than that reported in literature (2.4 to 5.8%)48. Previous studies already established an influence of the rearing substrate on the mineral content of BSFL. Moreover, Newton and colleagues48 reported higher calcium concentration for BSFL reared on poultry manure in comparison to the concentration we obtained from BSFL reared on a similar substrate. Such a variability could be further justified by the fact that the outer layer of the larvae’s skin releases a deposit of calcium carbonate (CaCO3) which may lead to the high calcium and ash content50. However, while the BSFL calcium levels we detected varied among the three organic waste streams tested, phosphorus levels remained unaffected in our study. The again, Newton and colleagues41 reported higher levels of phosphorus in BSFL reared on poultry manure than those reared on swine manure. This variability could also be attributed to the influence of the rearing substrate on the mineral content of BSFL.In addition to CP, other nutritional components of livestock feed can greatly enhance the quality of animal production, including several minerals and vitamins including calcium, magnesium, phosphorus, copper, cobalt and vitamins A or D. For example, calcium and phosphorous play important roles in x physiological functions of animals including muscle mass reductions, neuro-signaling, enzymatic activity, metabolic reactions, construction of proteins, maintenance of osmotic and acidic- alkaline equilibria, construction of membranes etc.51. Vitamins contribute essentially to the development of the immune system in animals while also helping in the digestion of other nutrients for energy production51. We did not observe any influence of the rearing substrates on vitamins in the BSFL. Though gamma and alpha tocopherol and provitamin D3 were detected in all BSFL samples in our study, Finke and colleagues46 and St-Hillarie and colleagues42 reported relatively higher levels of alpha tocopherol and provitamin D3. In addition, they found more vitamins including biotin, folic acid, vitamin A, and niacin in their studies with BSFL.Animal feed needs to contain sufficient quantities of vitamins to facilitate the development of proper and healthy body functions52. Though the amino acid profile of soymeal is generally of a better quality than that of other plant-based feeds, it is still deficient in lysine, methionine, threonine and valine when compared to an animal based protein source52. Previous studies established that the amino acid profile of several edible insects including the yellow mealworm, common housefly, and BSF is comparable to that of soybean meal with methionine or methionine and cysteine and sometimes arginine as the most limiting essential amino acids for growing swine and broilers53. We could show that BSFL reared on the three tested organic waste streams had a higher quality amino acid profile than the FAO54 standard amino acid profiles reported for soybean and sunflower meal. The BSFL methionine levels in our study even surpassed that of fishmeal reported by FAO54 and corresponded with the recommended range for broiler chickens as per the standards of the National Research Council (NRC)55. Similarly, the range of methionine levels (6.4 to 7.9%) in BSFL detected in our study surpassed those of methionine (0.7 to 0.9%) of BSFL reared on various organic waste streams and previously reported in literature49. Moreover, we detected higher lysine levels in BSFL than commonly found in soybean and sunflower meals2 and ours were only slightly lower than in fish meal2. Moreover, lysine levels detected in our study (4.1%) were higher than the levels previously detected by Arango Gutiérrez and colleagues56, viz. 2.1% for BSFL reared on chicken manure. Thus, rearing BSFL on the three tested organic waste streams resulted in terms of amino acids profile in a high quality protein source that would be in this respect nearly at par with fish meal and clearly surpass many plant-based protein sources in livestock feed.Amino acids are essential for quality livestock production, especially their ability to break down other proteins and to produce energy58. USFAs are important for human growth, skin protection and can decrease the possibility of thrombosis formation59. Insect fat is abundant in USFAs and resembles chicken and fish in their level of unsaturation60; yet, FAs profiles considerably vary among different insect species61. Such variability may be associated with environmental conditions such as the insect’s age, sex or size and its digestion and enzymatic activities62. We obtained higher USFA in comparison to SFA contents confirming the high quality of the BSFL FA profile. Like Makkar and colleagues2 we observed that the FA profile is influenced by the rearing substrate, with SG in general resulting in higher FA levels than the other two tested organic waste stream materials. BSFL FA profiles were predominantly composed of lauric, oleic and stearic acids. This corresponds to previous studies done by Leong and colleagues63 Zheng and colleagues64 though their values of lauric acid were relatively high compared to our results. The total amount of USFA in BSFL is close to those in olive, or soya oils65. In particular, the concentration of lauric acid in BSFL-derived oil is considerably higher than in those derived from soybean, sunflower, and oil palm66. Lauric acid is known to react against lipid enveloped viruses, and many pathogenic bacteria and protozoa69.FAs are usually subdivided into either saturated fatty acids (SFA) or unsaturated fatty acids (USFA). High levels of SFAs in diets such as palmitic and myristic acid are not favorable because they raise the level of low density lipoproteins (LDLs) by suppressing the expression of LDL receptors70. Studies on insects like edible stink bugs confirmed the presence of alkaloids, flavonoids and steroids among other bioactive compounds72. Flavonoid rich feed are sometimes associated with pharmacological effects73. In our BSFL we found rutin, apeginin and luteolin, belonging to the flavone class, usually associated with fruit skins, red wine, buckwheat, red pepper and tomato skin75 and kaemferol and quercetin belonging to the flavonol class that are more prevalent in onion, red wine, olive oil, berries and grapefruits76. The presence of these flavonoids in the insects can probably be traced back to the different rearing substrates and were acquired through feeding.Flavonoids occur naturally in vegetables, fruits and medicinal plants. Flavonoids occur naturally in vegetables, fruits and medicinal plants. They perform significant biological actions by acting as antioxidants, anti-carcinogens, anti-allergens, anti-pathogens and growth promoters in different animal species77) we did not detect any aflatoxins or other mycotoxins in our BSFL. Mycotoxins are metabolic products of fungi with harmful poisoning effects to animals and humans. Monogastrics like pigs and poultry are highly susceptible to such contaminated feed78. Thus, to further promote insect-derived feeds, routine and rigorous testing schemes for toxins and heavy metals, both in the rearing substrates as well in the harvested BSFL, have to be implemented. Purschke and colleagues79 reported that BSFL accumulated heavy metals but not mycotoxins when fed with contaminated substrates, though their concentrations in BSFL tissues -apart from Cd and Pb- remained below the initial substrate concentrations. Therefore, in order to ensure feed and food safety from farm to fork, screening of both the substrates and the BSFL-derived feeds as for contaminants such as Cd and Pb is a necessity. In addition to toxins, microbiological contamination might be a matter of concern when it comes to the utilization of insects in feedstock production. Indeed, insects are often affiliated to various microbes such as bacteria, fungi and viruses which are part of their defense repertoire80. However, BSF have shown an extreme resistance to various environmental conditions through their ability to reduce and possibly suppress possible bacterial and fungal contaminations aided by chemical and biological agents they produce82. It is suggested that contact with wild insects and other sources of contamination should be avoided in order for properly maintained insect mass rearing facilities to remain free from pathogenic hazards80. Moreover, the possibility of insects to harbor parasites can be limited and contained through introducing and maintaining environmental conditions that are unfavorable to parasitic existence80. Therefore and on the basis of our current knowledge, biological risks associated with insect consumption can be minimized using simple hygienic measures such as appropriate processing methods, i.e. heating or freezing similar to measures that are applied during processing of poultry, pork and fish80.Contrary to previous studies which showed that insects are prone to accumulate toxins or heavy metals ingested through contaminated feed or water , in Nairobi, Kenya.The study was carried in the laboratories of the International Centre for Insect Physiology and Ecology  with strong access to daylight spectra and temperatures maintained at 28 ± 5 °C to encourage mating to occur. The flies are supplied with water to prolong their life. Corrugated cardboard and some SG are placed within the cage to attract adult females for egg laying.86. KW was a mixture of potato peeling, carrot remaining and peelings, rice and bread debris (contained no meat) collected from a local restaurant in Nairobi. SG was sourced from Tusker House, Kenya Breweries Ltd. in Nairobi after fermentation of barley in the beer production process. The substrates were chosen on the basis of their availability in Nairobi, with a view of their potential future use for large scale industrial BSFL production in Kenya and beyond in Africa. Metallic trays measuring 23 × 15 cm were used to contain 1 kg of the different substrates. Each substrate had six replications placed randomly in a wooden frame in a room. Five-hundred (500) neonatal BSFL were placed carefully on top of the substrate in each of the trays. During the rearing process, the temperature was maintained at 28 ± 2 °C and relative humidity (r.h.) at 65 ± 5%. Distilled water was sprinkled on the substrate to ensure 65–70% moisture content. All substrates were replenished weekly with fresh ones. After reaching the prepupal stage, the insects were harvested, washed with tap water, oven dried at 60 °C for 48 hours, crushed using a laboratory blender and then stored for subsequent analyses in a refrigerator at −20 °C. A sample of the BSFL was collected from each replication, pooled and a 200 g sample was used for the subsequent analyses.The tested substrates, i.e. chicken manure (CM), kitchen waste (KW) and brewers’ spent grain (SG), were all sourced locally. CM was collected form a broiler poultry farm in the greater Nairobi area and used one week after it was harvested. Though the use of manure in feeding farmed animals including insects is prohibited in the European Union (EU), feeding farmed insects with any type of substrate is not an issue as long as the end product is free of heavy metals, microbial and mycotoxins contaminantsAflatoxin B1, B2 G1 and G2, were purchased from Supelco , apigenin (>99%), luteolin (>97%), rutin hydrate (>94%), quercetin dehydrate (>98%), octadecanoic acid (>98.5%), glutamic acid, myristoleic acid, tetradecanoic acid, hexadecanoic acid, (Z)-9-hexadecenoic acid, linoleic acid, (Z)-9-octadecenoic acid, octadecanoic acid, -9,12-0ctadecadienoic acid, oleic acid, eicosanoic acid and amino acid standard solution (AAS 18) were purchased from Sigma-Aldrich .34. Dry matter was calculated by the weight difference between before and after drying the sample in the oven at 135 °C for two hours; ash was determined by heating the samples in a muffle furnace set at 600 °C for three hours while CP was determined using the Kjeldahl method and a nitrogen-to-protein conversion factor of 4.76 was used in the calculation of CP35. Diethyl ether was used as an extractant in the determination of crude fat using the Velp solvent extractor (SER 148/6). Acid detergent fiber (ADF) and neutral detergent fiber (NDF) were analyzed with a Velp fiber analyzer (FIWE 6)  using reagents described by Van Soest and colleagues87. All the analyses were done in duplicates.Prior to the proximate analysis and after reaching the prepupal stage, the insects were harvested, washed with tap water, oven dried at 60 °C for 48 hours, crushed using a laboratory blender and then stored for subsequent analyses in a refrigerator at −20 °C. Then, the BSFL samples were taken for proximate analysis using standard methods of the Association of Official Analytical Chemist88 and detailed in Musundire and colleagues89 Briefly, the BSFL samples were separately snap-frozen in liquid nitrogen and crushed into fine powder. The samples (2 g each) were extracted for 1 hour in ice cold 5 v/w 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.2, 2 mM dithiothreitol (DTT), 2.5% Polyvinylpyrrolidone (PVP), 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM benzamidine 0.1 mM phenylmethanesulfonylfluoride (PMSF) in a magnetic stirrer. The samples were filtered through KERLIX™ Gauze Bandage Rolls Sterile Soft Pouch 5.7 × 2.7 cm centrifuged at 8,000 rpm for 30 min at 4 °C to remove solid debris. Protein was precipitated between 45% and 80% (NH4)2SO4 and the pellet recovered by centrifugation at 21,000 rpm for 30 min at 4 °C. The protein pellets were desalted in 20 mM HEPES–NaOH pH 8 containing 2mMDTT using Sephadex G-25 gel filtration chromatography  to give 80.2 mg and 77.9 mg of proteins from processed and unprocessed insect samples, respectively. Ten (10) mg from each of the samples were separately transferred into a 5 ml micro-reaction vial into which 2 ml of 6 N HCl were added and closed after careful introduction of nitrogen gas. The samples were hydrolyzed for 24 hours at 110 °C. For tryptophan analysis, 10 mg from each of the samples were separately transferred into a 5 ml micro-reaction vial into which 2 ml of 6 N NaOH were added and then capped after careful introduction of nitrogen gas. The samples were hydrolyzed for 24 hours at 110 °C. After the hydrolysis, the mixtures were evaporated to dryness under vacuum. The hydrolysates were reconstituted in 1 ml 90:10 water: acetonitrile, vortexed for 30 s, sonicated for 30 min, and then centrifuged at 14,000 rpm and the supernatant analyzed using a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Qtof-MS) . The analysis was replicated three times.The method for protein extraction was adopted from Hamilton and colleaguesAfter drying the prepupae and pre-experiment substrates, they were crushed using a laboratory blender and then ashed and digested in 6 N HCl. An Atomic Absorption Spectrophotometer  was used to analyze the following minerals: phosphorus, potassium, calcium, magnesium, sodium, iron, copper, manganese, cobalt and zinc.90 and Musundire and colleagues89. Ten (10) g of every sample were snap frozen in liquid nitrogen, crushed into fine powder and extracted in 40 mL acetonitrile-water  for 30 min while sonicating. Each mixture was allowed to settle for 30 min and then 6 mL of each sample was filtered through a solid phase extraction cartridge Multisep® 228 AflaPat multifunctional columns . An aliquot (4 ml) of each cleaned extract was evaporated to dryness in a stream of nitrogen gas. The dried samples were re-dissolved in 400 μL methanol-water , vortexed for 1 min and then centrifuged at 14,000 rpm for 5 min prior to analysis using a Waters liquid chromatography coupled to quadruple time of flight mass spectrometry (LC-QtoF-MS) . Samples derived from different substrates were analyzed in five replicates.Samples of BSFL were analyzed for mycotoxins according to methods described by Cheng and Cappozzo89. Briefly, a methyl esterification reaction was performed on 5 g of each sample according to procedures outlined by Christie91. A solution of sodium methoxide in methanol was prepared to generate a concentration of 15 mg/ml. An aliquot of the solution (500 µL) was added to each ground insect sample, vortexed for 1 min and then sonicated for 5 min. The reaction mixture was incubated at 60 °C for 1 hour, thereafter quenched by adding 100 µL deionized water followed by vortexing for another 1 min. The resulting methyl esters were extracted using GC-grade hexane  and then centrifuged at 14,000 rpm for 5 min. The supernatant was dried over anhydrous Na2SO4 and then analyzed using gas chromatography coupled to mass spectrometry (GC/MS) . Fatty acids were identified as their methyl esters by comparison of GC retention times and fragmentation patterns with those authentic standards and reference spectra published by library-MS databases National Institute of Standards and Technology (NIST) 05, 08 and 11. Serial dilutions of the authentic standard octadecanoic acid (0.2–125 ng/µg) were analyzed by GC/MS in full scan mode to generate a linear calibration curve (peak areas vs. concentration) with the following equation [y = 7E + 06x − 4E + 07(R2 = 0.9757)], which was used for the external quantification of the different fatty acids.BSFL samples reared on the three different substrates were snap frozen in liquid nitrogen and then ground into a fine powder and analyzed for fatty acids following the procedures described by Musundire and colleagues89 The samples were separately crushed into fine powder in liquid nitrogen. Two-and-a-half (2.5) g of each sample were independently extracted in 50 mL methanol-water (80:20 v/v) by ultrasonication in a sonication bath  for 1 hour followed by filtration through a Whatman filter paper No. 32. The remaining residue was re-extracted twice, and the filtrate pooled separately. The extracting solvent was removed under reduced pressure at 40 °C using a rotary evaporator  to give 60, 80, 100 and 120 mg for KW, CM and SG, respectively. The extracts (5 mg) from each of the samples were re-dissolved in 1 mL water-methanol (95:5 v/v), centrifuged at 14,000 rpm for 5 min and the supernatant analyzed using LC-Qtof-MS . Five replicates were carried out with each replicate done on a different larvae sample.BSFL samples reared on the three different substrates were analyzed for flavonoids following the procedures described by Musundire and colleagues89. LC-Qtof-MS and GC-MS instruments were used in the analysis. The instrument conditions included:The instrument conditions were similar to those described by Musundire and colleagues−1. The injection volume was 1 µL.The chromatographic separation was achieved on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) I-class system . For amino acids analysis, the UPLC was fitted with an ACE C18 column  with a heater turned off and an autosampler tray cooled to 5 °C. Mobile phases of water (A) and acetonitrile (B), each containing 0.01% formic acid, was employed. The following gradients were used: 0 min, 5% B; 0–3 min, 5–30% B; 3–6 min, 30% B; 6–7.5 min, 30–80% B; 7.5–10.5 min, 80% B; 10.5–13.0 min, 80–100% B, 13–18 min, 100% B; 18–20 min, 100–5% B; and 20–22 min, 5% B. The flow rate was held constant at 0.7 ml min−1 for both analyses.For analyses of flavonoids and aflatoxin, a UPLC was fitted to a Waters ACQUITY UPLC BEH C18 column  heated to 40 °C and an auto sampler tray cooled to 15 °C. Mobile phases of water (A) and methanol (B), each with 0.01% formic acid, were employed. The following gradients were used for i) flavonoids: 0–0.2 min, 10% B; 0.2–3 min, 10–60% B; 3–5 min, 60–80% B; 6–8 min, 80% B; 8–9 min, 100% B; 9–10 min, 100% B; 10–10.5 min 100–10% B; and 10.5–12 min 10% B; ii) aflatoxin: 0–0.2 min, 10% B; 0.2–3 min, 10–90% B; 3–5 min, 90% B; 5–6 min, 90–10% B; and 6–7 min, 10%B. The flow rate was held constant at 0.4 ml minE) in positive mode. Data were acquired in resolution mode over the m/z range 100–1,200 for flavonoids: m/z 100–700 for amino acid analysis with a scan time of 1 s using a capillary voltage of 0.5 kV, sampling cone voltage of 40 V, source temperature 100 °C and desolvation temperature of 350 °C. The nitrogen desolvation flow rate was 500 L/h. For the high-energy scan function, a collision energy ramp of 25–45 eV was applied in the T-wave collision cell using ultrahigh purity argon (>99.999%) as the collision gas. A continuous lock spray reference compound +  = 556.2766) was sampled at 10 s intervals for centroid data mass correction. The MS was calibrated across the 50–1,200 Da mass using a 0.5 mM sodium formate solution prepared in 90: 10 2-propanol/water (v/v).The UPLC system was interfaced with electrospray ionization (ESI) to a Waters Xevo QToF-MS operated in full scan based on independent information acquisition  was used for data acquisition and processing. The elemental composition was generated for every analyte. Potential assignments were calculated using mono-isotopic masses with a tolerance of 10ppm deviation and both odd- and even-electron states possible. The number and types of expected atoms was set as follows: carbon < 100; hydrogen < 100; oxygen < 50; nitrogen < 6; sulphur < 61 (0.01–20 ng/μl), rutin, quercetin, luteolin, apigenin (1.8–181 ng/μl), and glutamic acid (0.01–10 ng/μl) were also analyzed by LC-Qtof-MS in MSE mode to generate linear calibration curves (peak area vs. concentration) with the following linear equations: aflatoxin B1 [y = 13738x + 6611.5 (R2 = 0.9571)], rutin [y = 5578.4x − 39094 (R2 = 0.9960)], quercetin [y = 4372.4x + 79607 (R2 = 0.9854)], luteolin [y = 13433x − 23256 (R2 = 0.9994)] apigenin [y = 10288x − 11117 (R2 = 0.9995)] and glutamic acid [y = 40137x − 1353.1 (R2 = 0.9999)] which served as the basis for the external quantification of the aflatoxin, flavonoids and amino acids.Serial dilutions of authentic standards of aflatoxin B−1 to 280 °C, and held at this temperature for 20.4 min. The GC was fitted with a HP-5 MS low bleed capillary column  . Helium at a flow rate of 1.25 ml min−1 served as the carrier gas. The mass selective deceptive was maintained at ion source temperature of 230 °C and quadrapole temperature of 180 °C. Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. A 1.0 µl aliquot of sample was injected in the splitless mode using an auto sampler 7683 . Fragment ions were analyzed over 40–550 m/z mass range in the full scan mode. The filament delay time was set at 3.3 min.Fatty acid methyl esters (FAMEs) were analyzed by GC/MS on a 7890 A gas chromatograph  linked to a 5975 C mass selective detector  by using the following conditions: inlet temperature 270 °C, transfer line temperature of 280 °C, and column oven temperature programmed from 35 to 285 °C with the initial temperature maintained for 5 min then 10 °C minStatistical Analysis System  was used for analyses. Collected data was subjected to Levene-test to test for normality, followed by one-way analysis of variance (ANOVA) using the general linear model (GLM) procedure to test for significant differences among the means. In case of significant F-values, Bon-Tukey was used to separate the means at p < 0.05.


Peripheral artery disease (PAD) is frequently co‐prevalent with coronary artery disease (CAD) and diabetes (DM). The study aims to define the burden of CAD and/ or DM in PAD patients at moderate stages and further to evaluate its impact on therapy and outcome.Study is based on health insurance claims data of the BARMER reflecting an unselected “real‐world” scenario. Retrospective analyses were based on 21 197 patients hospitalized for PAD Rutherford 1‐3 between 1 January 2009 to 31 December 2011, including a 4‐year follow‐up (median 775 days).In PAD patients, CAD is prevalent in 25.3% (n = 5355), DM in 23.5% (n = 4976), and both CAD and DM in 8.2% (n = 1741). Overall, in‐hospital mortality was 0.4%, being increased if CAD was present . Both, CAD and DM increased long‐term mortality . DM further increased long‐term amputation risk , whereas CAD  did not.In a greater perspective, the data identify also mild to modest stage PAD patients at particular risk for adverse outcomes in presence of CAD and/or DM. CAD and DM both are related with a highly increased risk of long‐term mortality even in intermittent claudication, and DM independently increased amputation risk. Representing about 10% of the entire German population the BARMER GEK covers approximately 8.7 million insured patients. All German hospitals are obliged to transfer data on diagnoses, co‐morbidities and complications to the health insurances in form of detailed and obligatory coding guidelines: German Modification of the International Statistical Classification of Diseases and Related Health Problems 10th Revision (ICD‐10‐GM). For hospital reimbursement, all diagnostic and therapeutic procedures have to be coded according to the German procedure classification . The German Diagnosis related Groups (G‐DRG) system is used for hospital reimbursement in Germany since 2004. Annual adaptations of the ICD‐10‐GM were accounted for in the study. After patients' discharge, one code for the main diagnosis must be selected reflecting the underlying cause for admission. For additive coding of secondary diagnoses, a vast number of supplementary codes can be chosen representing co‐morbidities and complications which were present or occurred during in‐hospital stay. Reimbursement is calculated on the basis of patients' extent of diagnoses and the procedures performed. In general, factors enhancing the reimbursement  are unlikely to be omitted, since only complete coding is the essential condition for correct reimbursement and therefore of economic importance for the hospitals. About 20% of the codes are controlled—and corrected if required—by specialized physicians (“Medizinischer Dienst der Krankenversicherung”) independently from health insurances and hospitals.The anonymized data of the BARMER GEK health insurance are accessed as previously describedWe analyzed a total of 21 197 in‐hospital patients with primary or secondary diagnosis of PAD at Rutherford grades 1‐3 . The analysis includes all patients with an index‐hospitalization between 1 January 2009 and 31 December 2011, including a follow‐up period of at least 24 months after discharge until 31 December 2012 .Within these Rutherford grades (ICD 10 I70.20 and I70.21), subgroups with co‐diagnoses of diabetes mellitus  and coronary artery disease  have been identified. Further, co‐diagnoses of hypertension, obesity, dyslipidemia, smoking, chronic kidney disease, chronic heart failure, and malignancies have been ascertained according to ICD‐10 coding. Additionally, complications specifically acute renal failure, acute myocardial infarction, ischemic stroke, infection, and sepsis as well as in‐hospital mortality have been assessed accordingly. A detailed specification of definitions and ICD‐10 codes is given in the supplements  and its results were displayed as HR with 95%‐CI, and cumulative event curves.Within these subgroups, specific (co‐)diagnoses and procedures are stated as absolute numbers (n) and percentages (%) of the respective sub‐cohort. Categorical variables were statistically compared by use of the chi‐square test. Continuous variables were shown as mean ± SD and were analyzed by the 3P < 0.001) and high prevalence of common cardiovascular risk factors , but not smoking . Previous ischemic stroke was particularly present in PAD patients with concomitant DM , whereas chronic kidney disease (CKD) was equally increased by DM (18.1%) or CAD (19.8%) to 31.2% in PAD with DM and CAD.A total of 21 197 patients with mild to moderate PAD (Rutherford classification: Ruth 1‐3) were included in the analysis. Of these, 5355 (25.3%) suffered from concomitant CAD, 4976 (23.5%) from concomitant DM, and 1741 (8.2%) from both CAD and DM. Baseline characteristics of PAD patients with regard to CAD and DM are presented in Table P < 0.001). In contrast, previous vascular surgical procedures  did not differ significantly in these subgroups (Table P < 0.001).Overall, 7.6% of all PAD patients at Ruth 1‐3 had undergone previous endovascular revascularization procedures (EVR). Particularly CAD was associated with increased previous EVR . Of all PAD patients, 75.3% received a revascularization procedure . EVR was performed at lower frequency in subgroups with CAD alone (52.8%), DM alone (50.6%) and at most by the combination of CAD and DM . Whereas concomitant DM increased the length‐of‐stay (4 days vs 3 days without DM), CAD severely increased costs of the index‐hospitalization . Multivariate binary regression analysis showed CAD alone to independently increase the risk of in‐hospital mortality having an odds ratio (OR) of 1.849 .The rate of severe in‐hospital complications was at low level with infection as the leading diagnosis .The long‐term mortality and amputation were assessed during the follow‐up period of up to 4 years . During follow‐up, amputations were performed in 664 patients (3.1%). PAD patients with DM had significantly higher amputation rates than patients without DM . Both, CAD  and DM  independently increased long‐term mortality with highest risk if combined  PAD patients died during follow‐up. Mortality rates highly increased with co‐diagnosis of CAD and/or DM .Third, long‐term mortality increased to 21% in PAD with DM and CAD compared to 9% in patients without during 4‐year follow‐up  points at challenging conditions for secondary prevention.Concomitant DM was present in 23.5%, CAD in 25.3%, and both CAD and DM in 8% of all hospitalized PAD patients at Rutherford 1‐3. These numbers correspond well with the data reported by the get ABI cohort study on low‐stage PAD in the ambulatory sector .4.1DM was associated with previous amputation in already 1% of claudicants (compared to 0.2% in PAD patients with CAD), reflecting the relatively high risk of foot ulceration even in preserved perfusion of the limb .Although the actual need for revascularization in mild to modest stage PAD may be in principle questioned,P = 0.029) and follow‐up period  in line with current knowledge.In‐hospital mortality was generally low (up to 1.1%), however presence of CAD was independently associated with increased death during in‐hospital (OR 1.849; Nevertheless, given the fate of claudicants with an average 4‐year‐mortality rate of 11.5% in the overall cohort (15%‐30% at 5‐years in the literatureNot only that recognition of early stage PAD remains a matter of particular significance but also is consequent treatment of risk factors and timely prevention.4.2Strengths of our study is its large size of 21 197 unselected PAD patients reflecting “real‐life scenario” including a 4‐year follow‐up period. Diagnoses and procedures are based on encoded data using the German DRG and OPS code. Despite generally high data integrity and validity, inaccuracy within the limits of the encoding systems may have occurred. PAD diagnosis in routine‐data may hypothetically also include asymptomatic PAD since these are not clearly to be separated from mild intermittent claudication by ICD‐10 GM 2011. However, encoding asymptomatic PAD did not lead to higher reimbursement category as potential inducement for over‐coding. Further, DM and CAD may be under‐diagnosed in early PAD stages. These under‐recognitions would result in an underestimation of the presented hazards.Finally, information on underlying reasons for therapeutic decisions, the technical success of the procedures or concomitant medication is not included in the data set.5PAD patients are already at mild to moderate stage of disease at particular risk of death and amputation in the presence of CAD and/or DM. Future research is needed to clarify the benefits from early and consistent risk factor control in these patients. Particularly the role of endovascular therapy at early PAD stages with cardiovascular risk constellations should be addressed by future research to effectively prevent amputation.6Our study revealed a highly adverse impact of concomitant DM and CAD on the fate of PAD patients even at subclinical to moderate stages of disease. CAD was related with increased risk of short‐ and long‐term mortality, whereas DM highly increased amputation risk against the background of relatively low performance of revascularizations.The authors PS, NM, CE, FL, KG, MM, and EF have no conflicts of interest. JW reports personal fees and non‐financial support from Biotronik, Boehringer Ingelheim, Daiichi‐Sankyo as well as personal fees from Bayer Vital, MSD, Berlin‐Chemie, and Vifor outside the submitted work. HR reports personal fees from NovoNordisk, Pluristem, Daichii, BMS, DiaPlan, MedUpdate, and Pfizer outside the submitted work.PS and EF were responsible for data analysis and interpretation and wrote the manuscript. NM and HR advised on study design and provided careful review of the manuscript. CE was responsible for primary data analysis. FL, KG, MM and JW provided careful review of the manuscript and data interpretation. All authors read and approved the final manuscript.Table S1. ICD‐10 GM diagnostic codesTable S2. OPS procedure codesClick here for additional data file.


The prognostic effect of sidedness in colorectal cancer has been evaluated in numerous prospective and retrospective cohorts. Most of these have reported overall survival data; there is scant relapse‐free survival data in early stage disease. This study aimed to determine the effect of tumor sidedness in survival in early stage and relapsed colon cancer.Patients with stage I‐III colorectal cancer were identified from the BC Cancer Agency Gastrointestinal Cancer Outcomes Unit. Survival analysis by stage and sidedness was compared with the log‐rank test. Baseline characteristics were controlled by multivariate Cox‐proportional hazard models. In relapsed patients, bevacizumab and EGFR inhibitor (EGFRI) treatments were included and tested for interaction.Among 5378 patients with stage I‐III colon cancer, patients with right‐sided stage II tumors experienced better relapse‐free survival compared with those with left‐sided tumors; right‐sidedness was not prognostic for RFS in stage III disease. When survival was considered in patients who relapsed, right‐sided tumors had inferior survival after relapse in both stage II and stage III tumors. At relapse, right‐sided outcomes were inferior regardless of biologic therapy. An interaction test revealed a significant association between sidedness and survival with EGFRIs.In this large, population‐based study, right‐sided presentation has a significant prognostic impact: in early stage, right‐sidedness is favorably prognostic among stage II tumors and not prognostic in stage III disease. After relapse, right‐ sidedness is associated with an inferior prognosis, regardless of initial stage of presentation. Colon tumor sidedness is independently prognostic and may be considered in treatment assignment for both early stage and advanced disease. ACadjuvant chemotherapyCCacolon cancercfcell freeCIconfidence intervalCIMPCpg Island Methylator PhenotypeCMSconsensus molecular subtypesCTChemotherapyDFSDisease Free SurvivaldMMRdefective mismatch repairECOGEastern Cooperative Oncology Group (ECOG)EGFREpidermal Growth Factor ReceptorGICOUGastrointestinal Cancer Outcomes Unit (GICOU)HRhazard ratioIQRinter quartile rangeLLeft‐SidedLVIlymphovascular invasionMMRmismatch repairmOSmedian overall survivalNRNot reachedORRoverall response rateOSoverall survivalpMMRpreserved mismatch repairPFSprogression‐free survivalPNIperineural invasionRright‐sidedRFSrelapse‐free survivalSARsurvival after relapsewtwild‐type1Multiple studies have demonstrated differences between right and left‐sided colon cancer (CCa) in terms of clinical characteristics, pathology, and prognosis. Patients diagnosed with right‐sided CCa are more likely to be female, older, and have less acute presentation and more locally advanced tumors.In the stage IV or relapsed setting, right‐sided colonic tumors have undeniably worse outcomes when compared with left‐sided ones. Subgroup analyses of randomized clinical trials consistently show inferior outcomes in right‐ versus left‐sided tumors.P < 0.001], and this effect was independent of stage.The prognostic value of sidedness is much less conclusive in the early stage I‐III setting. In a meta‐analysis of 1 437 846 patients, left CCa conferred a favorable pooled prognostic effect on OS . Lymphatic or vascular invasion, and less than 12 nodes harvested, were each independently associated with worsened survival. Neither the receipt of adjuvant chemotherapy nor tumor grade influenced RFS among patients with stage II tumors. For stage III tumors, a stratified multivariate Cox model was used to allow for tumor grade and adjuvant chemotherapy with nonproportionality , P < 0.001].Overall survival analyses were conducted among the patients in the original cohort who developed a local, regional, or distant relapse Tables  and 4. R3.4KRAS wild type (wt) tumors  P < 0.0001]. A test of interaction did not reveal any association between bevacizumab therapy and sidedness (P = 0.195). Left‐sided, KRAS wt patients treated with EGFRI's [HR 0.37 (95% CI 0.22‐0.63)] and without EGFRIs [HR 0.72 (95% CI 0.64‐0.80)] also had superior outcomes ; however, a test of interaction revealed a significant interaction between sidedness and EGFRI therapy and OS (P = 0.017).Kaplan Meier median OS estimates of patients with relapsed disease are shown, based on tumor sidedness and receipt of bevacizumab , and other studies have demonstrated a similar effect .9‐0.91, aA significant finding of the current study was the lack of prognostic effect of sidedness among stage III patients, which calls into question whether sidedness is a pertinent consideration in the adjuvant treatment of node‐positive tumors. Results of a prior prospective trial of stage III CCa also demonstrated the lack of prognostic effect of sidedness on RFS,BRAF signature scores and contributed to inferior outcomes even among tumors that were microsatellite‐stable, BRAF, and KRAS WT, and may partially explain the poor survival of patients with right‐sided tumors independent of known prognostic factors.In the advanced setting after relapse, sidedness was a strong prognostic factor, with right‐ sidedness associated with an HR of 1.34 (95% CI 1.07‐1.68) in stage II and 1.31 (95% CI 1.14‐1.50) in stage III patients. The size of the prognostic effect was similar to that seen in stage IV disease in SEER cohorts, where HRs for right‐sided presentation were reported as 1.22 (95% CI 1.16‐1.28)KRAS WT left‐sided EGFRI‐treated tumors experiencing a significantly improved survival versus those with right‐sided tumors. Similar observations were made in randomized clinical trials. Post‐hoc analyses of multiple trials of first, second, and third line prospective trials of EGFRI therapy,The current study demonstrated the prognostic effect of sidedness in the advanced stage irrespective of bevacizumab or EGFRI therapy. However, a test of interaction demonstrated a significant association between sidedness and EGFRI therapy, with 4.1The major limitation of this study, which is one shared with numerous population‐based studies, is the lack of detailed molecular marker status. While sidedness is prognostic, it is very likely a surrogate for differences in the distribution of molecular factors or cancer subtypes between the right and left side of the colon. Such biomarkers would likely offer a more precise means to define prognosis and predict response to planned therapy than sidedness does. As All‐RAS and BRAF testing only became standard in advanced disease after 2013, this data is not available in this study which included only patients until 2009. Similarly, MSI status was not prospectively collected for the patients in this cohort. Nevertheless, multiple studies have demonstrated the prognostic effect of sidedness in the advanced setting that is independent of tumor genotype and Consensus Molecular Subtypes (CMS).5The study shows that right‐sidedness confers a favorable prognosis to stage II CCa but is not prognostic of relapse in stage III colon cancer. Right‐sidedness is a strong adverse prognostic factor among patients who relapse and may influence the selection of therapy. While more accurate biomarkers are awaited, sidedness should be considered as a relevant stratification factor in studies of both early and advanced disease, as imbalances between study arms can significantly affect study outcomes. Previously validated prognostic variables remain relevant and should continue to be incorporated in multivariate analysis and reflect the complexity of CCa disease biology.All of the authors contributed to the conception, data analysis, and writing of this paper.Conceptualization: Kennecke, Yin, Davies, Cheung, Lee‐YingData Curation: Kennecke, Yin, Speers, Cheung, Lee‐YingFormal Analysis: Kennecke, Yin, Speers, Cheung, Lee‐YingMethodology: Kennecke, Yin, Cheung, Lee‐YingWriting – review and editing: Kennecke, Yin, Davies, Speers, Cheung, Lee‐YingWriting –original draft: Kennecke, Yin, Davies, Cheung, Lee‐YingThis work was supported by the BC Cancer Foundation (BCCF). The BCCF did not provide any oversight nor did it have any influence in how the research was conducted.None of the authors have any conflicts of interest related to this paper. H.F.K. has served in an advisory role to Amgen Canada and Ipsen Canada. J.M.D. has served in an advisory role to Amgen, Bayer, Ipsen, and Spier. R.L.H. has served in an advisory role to Bayer and BMS. Y.Y., C.H.S., and W.Y.C. have no conflicts of interest to report.


HV decreases the arteriolar diameter of intracranial vessels, raising the risk of cerebral ischemia. The aim of this study was to characterize the effects of moderate short-term HV in patients with severe TBI by using concomitant monitoring of cerebral metabolism, brain tissue oxygen tension  probes during ICU admission within 36 h after injury were included in our study. Data collection and TCCD measurements were performed at baseline (A), at the beginning of moderate HV (C), after 50 min of moderate HV (D), and after return to baseline (E). Moderate HV was defined as arterial partial pressure of carbon dioxide 4–4.7 kPa. Repeated measures analysis of variance was used to compare variables at the different time points, followed by post hoc analysis with Bonferroni adjustment as appropriate.This prospective trial was conducted between May 2014 and May 2017 in the surgical intensive care unit (ICU) at the University Hospital of Zurich. Patients with nonpenetrating TBI older than 18 years of age with a Glasgow Coma Scale (GCS) score < 9 at presentation and with ICP monitoring, PbrO2 showed a statistically significant reduction but remained within the normal range.Eleven patients  with an initial median GCS score of 7 (IQR 3–8) were enrolled. During HV, ICP and mean flow velocity (CBFV) in the middle cerebral artery decreased significantly. Glucose, lactate, and pyruvate in the brain extracellular fluid did not change significantly, whereas PbrOModerate short-term hyperventilation has a potent effect on the cerebral blood flow, as shown by TCCD, with a concomitant ICP reduction. Under the specific conditions of this study, this degree of hyperventilation did not induce pathological alterations of brain metabolites and oxygenation.NCT03822026. Registered on 30 January 2019. This results in vasoconstriction of the cerebral arterioles [Hyperventilation-induced arterial hypocapnia (HV) has been used for decades in neuroanesthesia and neurointensive care, especially in the management of patients with severe traumatic brain injury (TBI) and concomitant elevated intracranial pressure (ICP). Increased alveolar ventilation induces a dose-response decrease in arterial partial pressure of carbon dioxide . Informed consent was obtained from the patients’ next of kin prior to study enrollment and/or from the patients after ICU discharge.2, and/or microdialysis probes, undergoing invasive mechanical ventilation with fraction of inspired oxygen < 60% and positive end-expiratory pressure < 15 cmH2O. Exclusion criteria were decompressive craniectomy, pregnancy, preexisting neurologic disease, previous TBI, acute cardiovascular disease, severe respiratory failure, acute or chronic liver disease, sepsis, and failure to obtain satisfactory bilateral TCCD signals. Patients with persisting hypovolemia or hemodynamic instability despite previous fluid resuscitation  were excluded. All patients were treated according to a cerebral perfusion orientated protocol aiming to achieve cerebral perfusion pressure (CPP) > 70 mmHg, ICP ≤ 20 mmHg, and PbrO2 > 15 mmHg. PaCO2 was targeted at 4.8–5.2 kPa.Inclusion criteria involved patients (≥ 18 years of age) with nonpenetrating head injury, with initial GCS score < 9 prior to sedation and intubation, extended neuromonitoring with ICP, PbrO2), end-tidal CO2 (etCO2), and PbrO2 were continuously monitored. ICP was measured with a fiberoptic device  and PbrO2 with a Clark-type microcatheter .MAP, ICP, CPP, arterial oxygen saturation  was performed using a catheter  with a membrane length of 10 mm and a molecular mass cutoff of 20 kDa, which was perfused by a microinjection pump  with artificial cerebrospinal fluid at a flow rate of 0.3 μl/min. Samples were collected every hour and immediately analyzed for glucose, lactate, and pyruvate with a bedside MD analyzer . Sampling was performed at 1-h intervals in order to allow the detection of sufficient concentration of analytes in the microdialysate. ICP, PbrOTCCD examinations of the middle cerebral artery (MCA) were performed bilaterally through the transtemporal acoustic window by two experienced physicians  according to standard techniques , 15 usin2 of 0.7 kPa  as a baseline measurement and 1 h (D + 1 h) and 2 h (D + 2 h) after D to detect possible changes induced by moderate HV. Arterial blood gas tests (ABG) were obtained at points A, C, D, and E to monitor the changes of pH and PaCOF-ratio of the ANOVA reached a critical level (corresponding to p < 0.05), post hoc analysis with Bonferroni adjustment was used. All tests were performed two-sided, and p < 0.05 was considered statistically significant. Statistical analysis was performed using Stata version 12.1 software .Data were analyzed to produce mean ± SD or median with IQR, unless otherwise indicated. Repeated-measures analysis of variance (ANOVA) was used to compare all variables. When the During the study period, 628 patients with TBI were admitted to the surgical ICU. A breakdown of reasons for exclusion is presented in Fig. On average, the Injury Severity Score (ISS) was 31 ± 11, and the Simplified Acute Physiology Score (SAPS) II 24 h after TBI was, on average, 48 ± 9. The study was performed, on average, 23 h after TBI.2 during the test and other respiratory variables are shown in Table Changes in PaCOp < 0.0001) during C, and remained stable during D . On return to normoventilation (E), ICP increased significantly to 14 ± 6 mmHg (p = 0.003 compared with D) and was similar to A (p = 0.06).MAP and HR Table  did not p = 0.0002) and to 66 ± 17 cm/s in the left MCA (p = 0.006). CBFV did not change significantly during D  compared with C. During E, CBFV was 77 ± 28 cm/s  and 75 ± 24 cm/s  at the right and left sides, respectively.TCCD measurements in the right and left MCAs were analyzed separately. CBFV was 81 ± 23 cm/s and 80 ± 22 in the right and left MCAs, respectively. During C, CBFV decreased to 69 ± 21 cm/s in the right MCA , increased significantly during D , and remained stable during E .PI was 1.03 ± 0.17 and 0.96 ± 0.15 in the right and left MCAs, respectively. During the study, PI did not change significantly in the right MCA , decreased during D , and remained stable during E (30 ± 8 p = 0.90 compared with D)  to investigate this.The aim of this study was to detect possible adverse effects of moderate HV in patients with severe TBI during the acute phase postinjury. To our knowledge, this is the first study using a multimodal monitoring set  ratio was not indicative of an energy crisis.On the basis of our results, there was a PbrO2 of 31–32 mmHg for the first 5 study days. Additionally, HV was induced prophylactically, regardless of ICP.In the last 30 years, HV has been fiercely debated. After initial widespread use , the eff2 reduction from 4.8 to 3.9 kPa. Diringer et al., on the contrary, using an analogous PET technique, did not disclose any alteration of oxygen metabolism in two groups of patients with TBI managed with different degrees of HV [More accurate studies of pathophysiological changes induced by HV have been performed by two authoritative groups. Coles et al. measured a significant reduction of CBF with PET scans and an increase in the volume of severely hypoperfused tissue following HV  with a Pes of HV . They co2 probes explore only small areas of the brain; therefore, we cannot exclude concomitant changes in other brain regions [Our data questioned if HV, even when effective in reducing CBF and ICP, induced measurable changes compatible with true ischemia (defined as a CBF insufficient for preserving adequate tissue oxygenation and metabolism). For a fair comparison with the existing evidence, however, the study limitations should be acknowledged. First, the small sample size limits the generalizability of the findings. More accurate analyses, for instance looking at sex differences in vasoreactivity or association between PI and outcome, were clearly not feasible , 22. Sec regions , 25.2 probes were placed, in the majority of the cases, in the most injured hemisphere of the brain. Interestingly, the cerebral glucose at baseline was normal, whereas the L/P ratio at baseline was markedly increased and pathological [2 should not have been compromised [In our study population, however, the MD and the PbrOological , reflectpromised .2, but within a physiological range, and does not significantly change the cerebral energy metabolism. Further investigations of prolonged and more severe HV, in the context of intracranial hypertension and in a larger study population, are needed to characterize the risks and benefits of HV in severe TBI.Our findings add evidence to the belief that a moderate degree of short-term HV is effective in reducing ICP through CBFV modifications as an estimation of CBF. Furthermore, a moderate degree of HV reduces PbrO


Sperm DNA integrity is considered essential for successful transmission of the paternal genome, fertilization and normal embryo development. DNA fragmentation index  has become a key parameter in the swine artificial insemination industry to assess sperm DNA integrity. Recently, in some elite Norwegian Landrace boars (boars with excellent field fertility records), a higher level of sperm DFI has been observed. In order to obtain a better understanding of this, and to study the complexity of sperm DNA integrity, liquid preserved semen samples from elite boars with contrasting DFI levels were examined for protamine deficiency, thiol profile and disulphide bonds. Additionally, the DNA methylation profiles of the samples were determined by reduced representation bisulphite sequencing (RRBS).n = 18 ejaculates). Upon liquid storage, the levels of total thiols and disulphide bonds decreased significantly, while the DFI and protamine deficiency level increased significantly. The RRBS results revealed similar global patterns of low methylation from semen samples with different levels of DFI . Differential methylation analyses indicated that the number of differentially methylated cytosines (DMCs) increased in the low-high compared to the low-medium and the medium-high DFI groups. Annotating the DMCs with gene and CpG features revealed clear differences between DFI groups. In addition, the number of annotated transcription starting sites (TSS) and associated pathways in the low-high comparison was greater than the other two groups. Pathway analysis showed that genes (based on the closest TSS to DMCs) corresponding to low-high DFI comparison were associated with important processes such as membrane function, metabolic cascade and antioxidant defence system.In this study, different traits related to sperm DNA integrity were investigated (To our knowledge, this is the first study evaluating DNA methylation in boar sperm cells with different levels of DFI. The present study shows that sperm cells with varying levels of DNA fragmentation exhibit similar global methylation, but different site-specific DNA methylation signatures. Moreover, with increasing DNA fragmentation in spermatozoa, there is an increase in the number of potentially affected downstream genes and their respective regulatory pathways. Sperm cells have a different chromatin structure compared to somatic cells. In somatic cells, DNA is wrapped around histone proteins, which allows DNA condensation. In contrast, during spermatogenesis histone proteins are, to a great extent, replaced by protamines coupled by disulphide bridges, a process that facilitates tight packaging of DNA in the sperm nucleus . Sperm cEpigenetics is a phenomenon where a series of events such as DNA methylation, histone post-translational modification (PTM) and close association with small RNAs, independently or in concert, control gene expression, without altering the DNA sequence . During Both sperm DNA methylation and fragmentation have been reported to correlate with fertility and field performance in different livestock. For instance, it has been shown that site-specific sperm DNA methylation status correlates with infertility in boars  and reprReduced representation bisulphite sequencing (RRBS) allowing the study of methylation profiles at single-base resolution, while experiment costs are kept low . RRBS isThe liquid diluted boar semen produced for pig production in Norway is recommended to be used within 96 h upon collection. However, due to factors such as long-distance transport and a long shipment time, the semen is often stored for 48 to 96 h prior to artificial insemination (AI) . RecentlAn overview of sperm DNA integrity parameters is presented in Table Furthermore, potential correlation between DFI and other sperm DNA integrity parameters was investigated Table . AlthougAn overview of the RRBS libraries and their basic statistics is provided in Table 2: 0.0046, p-value: 0.7877).CpG coverage and methylation levels for a representative sample are presented in Fig. 10  methylation levels, the samples are distributed in two clusters small (4 samples) and large (14 samples). However, the samples from different DFI groups don’t appear to cluster together  with varying levels of methylation ranging from 0 to 100% in the low-medium (LM) and low-high (LH) groups, respectively  to filtered DMCs and their corresponding gene information were extracted. This resulted in a greater number of TSSs in the hypo groups compared to the hyper groups, including 98, 43, 333 and 70 TSSs for LM hypo, LM hyper, LH hypo and LH hyper, respectively Fig. a. Previop-value for multiple testing in pathway analysis, genes with nearest TSSs to filtered DMCs in the LM hypo group were significantly associated with acetylation and phosphorylation pathways. A total number of 20 important biological process including acetylation, phosphorylation, membrane function, metabolic cascade and antioxidant defence system were connected to TSSs extracted from the LH hypo comparison  showed clear group differences, as none of filtered DMCs were present in the promoter and exons in the LM comparison compared to the LH comparison Fig. . It is wSperm DNA integrity has been linked with several important biological functions in sperm cells and offspring. We have recently shown that litter size in both Norwegian Duroc and Landrace breeds is negatively correlated with DFI . FertiliBriefly, results in this study show how different DNA integrity parameters in boar sperm cells correlate with each other and to what extent DNA methylation is different in sperm cells with different levels of DFI. Our results demonstrate that DFI is only slightly correlated with other sperm DNA integrity parameters. Based on RRBS analysis, the number of DMCs, as well as the number of linked pathways in the LH group, were higher compared to the LM group. This suggests that samples with higher DFI could potentially have a greater range of affected biological pathways including those involved in embryo developmental processes.In the current study, sperm phenotypic traits linked to DNA integrity including DFI, protamine deficiency, free thiols, total thiols, and disulphide bonds were analysed in boar semen both on the day of collection (Day 0) and upon 96–108 h storage (Day 4). As DFI exhibited the greatest contrast between samples compared to other phenotypic traits, samples selected and grouped based on their DFI level on Day 4, were subjected to RRBS.n = 28) were collected from seven Norwegian Landrace (NL) boars (240–570 days old), using the “gloved hand” technique. The boars were housed and cared for according to international guidelines and regulations for keeping pigs in Norway, at Norsvin artificial insemination (AI) station, in Hamar, Norway. After semen collection, ejaculates with motility above 70% and morphological abnormalities below 20%, were further diluted to 28 × 106 cells/ml using either the commercial extender Tri-X-cell®  or Androstar® Plus extender . According to Norsvin recommendations, semen doses can be stored at 18 °C up to 4 days (96–108 h), prior to AI and in Norway most of the semen are used at Day 3 and 4. Aliquots (1 ml) from both Day 0 and Day 4 semen samples were frozen at − 80 °C until analyses. In order to mimic the status on the day of use in the herds and to consider the worst-case scenario, only semen samples from Day 4 with high, medium and low DFI contrasts were included for RRBS analyses.Ejaculates . This procedure was followed by denaturation of sperm DNA for 30 s by adding an acid solution  and subsequently the samples were stained with acridine orange (AO) staining buffer . For each ejaculate, two technical replicates were analysed. Samples were protected from light and incubated at room temperature for 3 min prior to analysis of 5000 cells per technical replicate by a flow cytometer equipped with a blue laser (488 nm) . The flow cytometry instrument was AO-saturated prior to analysis, by running the AO equilibration solution (1.2 ml AO staining solution and 400 μl acid detergent solution) for 5 min. Green and red fluorescence signals were collected with a 525 nm band pass and a 670 nm long pass filter, respectively. To control laser stability, mean green and red fluorescence signals were re-set to 425 ± 5 and 125 ± 5, respectively, using a boar semen reference sample with known DFI at the start of the analysis and after every fifth sample. According to SCSA principle, after AO staining, double- and single-stranded DNA, emits green and red fluorescence, respectively. The percentage of red and green fluorescence was determined using the Cell Lab QuantaTM SC MPL analysis software . Based on the ratio of red/(red + green), the DFI was calculated.DFI was analysed using sperm chromatin structure assay (SCSA) according to Evenson et al. (2001)  and Boe-6 cells/ml) and washed with PBS by centrifugation . The sperm pellet was resuspended in 100 μl McIlvaine’s buffer  containing 0.25 mg/ml CMA3. Prepared samples were protected from light and incubated for 20 min at 37 °C. Stained sperm samples were washed in 500 μl PBS by centrifugation  before the pellets were resuspended in 500 μl PBS containing 4 μl propidium iodide . All samples were analysed in a flow cytometer  equipped with a blue laser (488 nm). The sperm cell population was gated using FSC and SSC and sperm cells were further identified by PI positive signal collected via 586/42 bandpass filter. The CMA3 fluorescence from gated cells was collected through a 528/45 bandpass filter after excitation with a violet laser (405 nm).The level of protamine deficiency in boar spermatozoa was assessed using Chromomycin A3 , according to the method described by Zubkova et al. (2005)  with min6 cells/ml) and divided into two tubes, each containing 1 × 106 cells. Tube 1 was loaded with 1 mM/l of 1,4-dithiothreitol  and was incubated for 10 min at 37 °C while no DTT was added to tube 2. Both tubes were centrifuged for 10 min at 300 x g and the pellets were resuspended in 100 μl PBS containing 0.5 mM of mBBr solution. This procedure was followed by incubating both tubes for 10 min at 37 °C, protected from light. Stained spermatozoa were washed in 500 μl PBS and centrifuged for 10 min at 300 x g. The pellets were resuspended in 500 μl PBS and analysed with a FACSVerse flow cytometer (BDBiosciences) as described above.Free and total thiols, as well as disulphide bonds in boar spermatozoa, were analysed using monobromobimane , as described by Zubkova et al. (2005)  and SeliIn order to calculate disulphide bonds, free thiols fluorescence signals (mBBr fluorescence from non-DTT treated samples) were subtracted from total thiols fluorescence signals (taken from the corresponding DTT treated samples), then the value was divided by two.RRBS library preparation and sequencing were performed according to Boyle et al. (2012) , with slDNA from Day 4 frozen semen samples was isolated using Maxwell 16 Benchtop DNA extraction instrument . Isolated DNA was quantified using Qubit dsDNA BR assay kit  and further diluted to 20 ng/μl in low TE media  at Biobank AS, Hamar.MspI and Taq α1 enzymes . Gap filling and A-tailing were conducted by adding 1 μl of Klenow fragment  as well as 1 μl of 10 mM dNTP mixture  into each digestion reaction well in a reaction plate. The samples were incubated  in a thermocycler without the heated lid and at the end, 300–500 bp fragments were further selected using SPRI AMPure XP beads . Thereafter, 2 μl of NEXTflex™ Bisulphite-Seq barcodes  was added into each well and ligation reaction was performed by incubating the adapter ligase mixtures at 16 °C overnight. Prior to bisulphite treatments, fragments were further size selected by adding 60 μl (2x) 20% PEG 8000/2.5 M NaCl  followed by incubation for 30 min at room temperature and the fragments were suspended in elucidation buffer. In order to evaluate the efficiency of adapter ligation as well as to determine the optimal PCR cycle number for later amplification, size-selected fragments were subjected to PCR amplification  with primers designed to bind with flank adapters. The optimal PCR cycles were found to be 13 and the PCR products were run on a 4–20% Criterion precast polyacrylamide TBE gel  and DNA bands were visualized by adding SybrGold  dye.Genomic DNA (400 ng) was digested overnight at 37 °C using Size-selected fragments (20 μl) were subjected to bisulphite conversion using the EpiTect kit  following the manufacturer’s protocol designated for DNA extracted from FFPE tissues.Bisulphite-converted DNA was cleaned up according to the protocol described in the QIAGEN EpiTect kit and . CleanedElucidated samples were sequenced using the Illumina HiSeq 2500 in the single end (1 × 100 bp) mode.Illumina adapter sequences and low-quality bases (below 20 bp and Phred score of 20) were trimmed from raw single end Illumina reads using Trim Galore! software (v 0.4.4) .Sus scrofa 11.1 genome [MspI digested RRBS genome was 3% of the total genome, which is an increase of 16% compared to the previous version [In this study, the recently published 1 genome  was used version . Clean a version . Bismark10) were considered for downstream analysis. Sperm samples with different DFI levels were divided into low (L), medium (M) and high (H) DFI based on our previous investigation [10 between low – medium (LM), low – high (LH) and medium – high (MH) groups. Six samples each from L, H, and M were allocated to every group. The “min.per.group” option was set to four in unite function [SAM sorted alignment files from Bismark were analysed using the methylKit package (v 1.6.1)  in Rstudtigation . For dowfunction . FurtherS. scrofa 11.1 assembly were downloaded from the UCSC table browser [BED files for Gene and CpG annotation for  browser . The DMCp-value was Benjamini corrected for multiple testing and was set to 0.05.In order to perform Gene Ontology (GO) analysis, extracted nearest TSS and gene ids to filtered DMCs, were submitted to DAVID Bioinformatics resources for functional annotation . Fisher’p-values were Bonferroni adjusted to 0.016 for total and free thiol as well as disulphide bonds. p-value for protamine deficiency and DFI was set to 0.05. Correlation between DFI, thiol profile and protamine deficiency as well as DFI and global CpG level was obtained via multiple linear regression model within the lme4 package and p-value was set to 0.05.Statistical analysis was performed in Rstudio, v 1.1.383 for windows. Linear mixed models within the lme4 package were established using phenotypic traits of sperm cells and preservation time as dependent and independent variables, respectively. In addition, individuals, animal age and season for the collection of semen as well as number of ejaculates were included as random effects. In order to minimize type I error, Results were plotted using GraphPad Prism . Volcano plots and Venn diagrams were constructed using Instant Clue (v 0.5.2 for Windows) and FunRich (v 3.1.3 for Windows) software, respectively , 50. PatAdditional file 1. CpG basic statistics for all samples. Figure Additional file 2. Differential methylation and downstream analyses for Medium-High (MH) group. Figure Additional file 3. List of closest TSS to filtered DMCs and pathway analysis results in Medium-High (MH) group.Additional file 4. Number of DMCs and DMRs after applying different methylation difference cut-offs.


Androgen hormone levels are strongly associated with obesity in adult mammals, especially with advanced age. We investigated androgen receptor inhibition on fat metabolism and long-chain fatty acid (LCFA) transport proteins in skeletal muscle during exercise.Male ICR mice were randomly divided into three groups: CON (control), EX (exercise), and EXIN (exercise + androgen receptor inhibition). EX and EXIN groups were trained on a treadmill five times a week. After 4 weeks, the fat metabolism of each group was measured using open-circuit calorimetry during 1 hour of exercise. After the metabolism measurement, the expression levels of LCFA transport proteins  were analyzed in skeletal muscle.P < 0.05). However, there was no different between the CON and EXIN group. Oxygen uptake and fat oxidation during exercise tended to be lower (12%) in the EXIN group than in the EX group. Total fat oxidation in the EXIN group was significantly lower during the initial 20-min (P < 0.003) and 40-min (P < 0.041) phases compared to that in the EX group. In addition, the level of FAT/CD36 protein in the EX and EXIN groups was approximately double that in the CON group . CPTI expression in the EX group was higher than that in the EX group (P < 0.0069) as well as in the CON group.Weight gain and final body weight were significantly lower in the EX group than in either the CON or EXIN groups. Conversely, food intake was significantly higher in the EX group than it was in the CON and EXIN groups. The total weight  of the abdominal adipose tissue were significantly lower in the EX group than in the CON and EXIN groups . Blocking androgen receptors can decreases the expression of CPTI in the skeletal muscle, which reduces fat metabolism. Thus, reducing sex hormones or suppressing the sensitivity of AR receptors can inhibit energy efficiency and fat metabolism by suppressing CPTI. Androgen hormones are negatively associated with the central obesity index in older adults .. AndrogAn androgen is any natural or synthetic steroid hormone in vertebrates that binds androgen receptors (AR) to regulate the development and maintenance of male characteristics . ARs, meARs are present in muscles and brown adipose tissues (BAT) that consume and expend energy . ARs areGuerrero J et al. subjected 9-week-old male CB17SCID mice to an AR inhibitor  and measured tumor volume and body weight at 2-to 3-day intervals for 4 weeks . The AR Endurance exercise increases capillary density, mitochondrial population, and the activity of the tricarboxylic acid cycle and other oxidative enzymes  . In addiThe direct effect of AR blockade is understood to be a decrease in resting metabolic rate and a concomitant increase in body weight . We prevTwenty-four male ICR mice were obtained from Orient Bio Inc.  and adapted to the laboratory housing conditions for 1 week. They were given free access to water and a non-purified commercial diet  containing crude protein (180 g/kg); crude fat (52 g/kg); crude fiber (52 g/kg); minerals (57 g/kg); and carbohydrates (368 g/kg). The protein, fat, and carbohydrate ratio (%) based on calories was 21:14:65. The gross and metabolizable caloric contents of the diet were 4.04 and 3.21 kcal/g, respectively.n = 8), EX , EXIN . Body weight and food intake were measured daily for 4 weeks. EX and EXIN groups underwent training by running on a treadmill five times per week for 4 weeks. The AR inhibitor enzalutamide  was dissolved in mixed solution (2% dimethyl sulfoxide in distilled water). Based on previous studies, AR inhibitor was administered orally every day for 4 weeks [At the age of 7 weeks, the mice were randomly divided into three groups: CON  for 3–4 weeks , 17.After 4 weeks of training, energy metabolism was measured during 1 hour of exercise using the training conditions of the final week . Two hours before the measurement, the mice were placed in exercise metabolic chambers with a volume of approximately 3 L to reduce stress. The flow rate was kept constant at 3 L/min and measured for 1 h. The energy metabolism during exercise was measured using an open-circuit device based on methods reported in previous studies .After metabolic measurement subjects were euthanized by sodium pentobarbital overdose. Skin was removed from the hind limbs and soleus muscle was extracted by established methods .The muscle (soleus) tissue samples (35 mg) were homogenized in 700 μL EzRIPA lysis buffer  using a mortar and TissueRuptor . The muscle lysates were mixed using a rotator for 2 h at 4 °C and then centrifuged at 12,000 rpm at 4 °C for 15 min. The protein concentration of the supernatant was determined using a GenDEPOT protein assay plus reagent kit  using bovine serum albumin (BSA) as the standard.Total protein (25 μg/lane) was separated using 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) at 80–110 V for 150 min and then transferred to a polyvinylidene difluoride (PVDF) membrane  at 100 V for 2 h. The membrane was blocked for 1 h at 25 °C with phosphate-buffered saline  containing 5% skim milk  and then washed three times  with PBS plus 0.1% Tween 20 (PBS-T) buffer. After an overnight incubation at 4 °C with primary antibodies against FAT/CD36 and CPTІ , the membranes were washed with PBS-T and incubated with an HRP-conjugated secondary antibody for 1 h at 25 °C .Immunodetection was carried out using an enhanced chemiluminescence (ECL) detection reagent . Quantitative analysis was carried out using the Image J program  including data from at least three independent experiments.Blood samples were collected after for 4 weeks. Plasma glucose was measured using commercial kits , the plasma FFA level using a non-esterified fatty acid kit , the plasma insulin level was determined with an enzyme-linked immunosorbent assay kit , and the plasma glycerol level was determined using the colorimetric assay kit  according to the instruction of the manufacturer.P < 0.05.Data are given as means ± standard deviation (SD). All statistical analyses were performed with SPSS version 19.0 software . Oxygen uptake, RER (respiratory exchange ratio), carbohydrate oxidation, fat oxidation, food intake, and body weight were analyzed by two-way repeated measures analysis of variance (ANOVA). One-way ANOVA was used to determine the changes in the sums of the energy metabolism during exercise, body weight, and food intake. Least squares difference (LSD) post-hoc analysis was conducted if significance was obtained. Differences were considered significant at P < 0.001, P < 0.001). However, the EXIN group underwent the same exercise intensity as the EX group but did not lose weight, gaining a similar amount as the CON group (P = 0.619). Nevertheless, food intake (in g/4 weeks and g/day) was significantly higher in the EX group than in the CON and EXIN groups . The total weight  of the abdominal adipose tissue were significantly lower in the EX group than in the CON and EXIN groups (P < 0.05). However, there was no different between the CON and EXIN groups. In additional, the mesentery fat was significantly higher in the EXIN group than in EX group (P < 0.05). However, there was no significant different between the EXIN and CON groups. Furthermore, The EX group tended to have less abdominal fat than other groups. On the other hand, the EXIN group showed similar fat weight as the CON group without exercise.Table P < 0.001) and 7% (P < 0.05) lower in EX and EXIN groups than in CON groups. In additional, plasma glycerol levels were 2.17-fold (P < 0.001) and 1.7-fold (P < 0.001) higher in EX group, respectively compared to respective CON and EXIN groups. However, no significant different between the CON and EXIN groups. Insulin levels were lower by 46 and 30% in EX and EXIN groups compared to CON group .Table P < 0.001), while interaction (P = 0.298) and group (P = 0.351) did not . Group-by-time interaction was also significant (P < 0.001) but group was not (P = 0.386) , interaction (P < 0.001), and group (P = 0.060)  Fig. a. It was60) Fig. b, while P < 0.001), interaction (P < 0.001), and group (P < 0.016) during the 1 h exercise . In addition, the EX group showed higher fat oxidation than the CON and EXIN groups after 40 min of exercise . However, the EXIN group experienced the same intensity as the EX group but did not produce a high fat oxidation, like the CON group.Fat oxidation was affected by time   .Western blot analysis was performed using protein obtained from the mouse skeletal muscle (soleus) samples. The FAT/CD36 protein level in the EX and EXIN groups was approximately double that of the CON group  showed significantly higher body weight, abdominal fat tissue weight  compared with the placebo treated exercise group. Our results suggest that the suppression of androgen hormone activity or lowering the sensitivity of AR can inhibit energy expenditure and fat oxidation by reducing CPTІ in skeletal muscle.P < 0.003) and after 20–40 min (P < 0.041). Furthermore, we found that body weight, abdominal fat  and plasma glycerol levels were significantly higher for the EXIN group compared to the EX group. Because both trained groups experienced elevated energy expenditure, the AR inhibited group may indicate lower energy efficiency and dependence on carbohydrate utilization during exercise. In additional, plasma insulin was found to be reduced in both in EX and EXIN groups due to exercise effects. However, FFA showed a significant decrease only in the EX group than in the CON group. This means that the triglycerides breakdown smoothly and FFA released into the blood is well used as energy source.In this study, we observed that oxygen uptake tended to be elevated during the initial exercise phase in the EX group, higher than the CON and EXIN groups. Interestingly, the EXIN group, which exercised with the same intensity during the same period, experienced decreased oxygen uptake during exercise. The EXIN group experiencing AR blockage also showed a significant decrease in fat oxidation  during the initial 0–20 min  increased energy consumption during 30 min of moderate intensity treadmill exercise . In addiP < 0.001, P < 0.0125). Continuous exercise has been reported to increase the expression of FAT / CD36 and CPTІ. These molecules transport fatty acids, mobilizing them for use as an energy source [P < 0.0069), while FAT/CD36 expression did not decrease even if ARs is blocked. This pattern was less pronounced in the CON (non-exercise) group. In present study, AR inhibition has not affected the expression of FAT/CD36 while decreasing the expression of CPT1. This seems to be a gene that, unlike CPT1, is not affected by androgen hormones and is increased independently through exercise. Meanwhile, the decrease in CPT1 in our study seems to be due to the activation of Malonyl-CoA. Malonyl CoA is a potent inhibitor of carnitine palmitoyl transferase (CPT-1), an enzyme that controls fatty acids transport into the mitochondrion [In this study, the FAT/CD36 and CPTІ protein levels were significantly higher in the EX group than in the CON group  compared with the CON (sedentary) and EX (exercise training with placebo). Values are presented as means ± standard deviations (n = 8).