This study was designed to investigate the current epidemiological situation of bovine viral diarrhea virus (BVDV) and rift valley fever virus (RVFV) infection of camels originating from Sudan “smuggler” and Egypt as part of our future plan for a national surveillance program in Egyptian provinces, which will aid in establishment of control strategy for animal diseases.This investigation was accomplished using serological diagnostic and molecular biology techniques. A total number of 200 blood samples were collected from camel  and were subjected for testing both BVDV and RVFV occurrence with different age and sex.Sixty-six of the 200 camels (33%) were positive for BVDV antibodies, and 44 (22%) for BVDV antigen (Ag), and 27 of the 200 camels (13.5%) were positive for RVFV immunoglobulin G (IgG) antibodies. On the other hand, the seroprevalence of BVDV for antibodies (47.5%), Ag (31.6%), and RVFV IgG antibodies (16.6%) was higher in camel originated from Sudan “smuggler” than of local breed which was 11.2% for BVDV antibodies and 7.5% for BVDV Ag, while it was 8.7% for RVFV IgG antibodies. The incidence of BVDV antibodies, Ag, and RVFV IgG antibodies was the highest in male, up to 9 years of age. The frequency of positive cases was significantly different according to the origin of samples and sex and age of camel for BVDV and RVFV. In addition, seven serologically positive samples for BVDV and five serologically positive samples for RVFV were submitted as a buffy coat for molecular detection by one-step – reverse transcription polymerase chain reaction (RT-PCR). The results demonstrated that three samples were positive for BVDV of camel originated from Sudan (smuggler), while no RVFV Ag was detected in all five samples. Sequencing and phylogenetic analysis of the amplicons obtained from positive RT-PCR samples (three samples) indicated 100% nucleotide homology with Sudan strain 2015 except only one (missense point mutation) by substitution of A to T at position 345 that changed the coded amino acids from T (Threonine) to S (Serine) at residue 115.Camels act as risk animals for the introduction of many infectious diseases from Sudan to Egypt, especially transboundary animal diseases, so strict quarantine measures should be taken during importation of live animals from Sudan to prevent the spread of such diseases. Camels are considered an important source for human food, transportation, and entertainment, especially in the Arabian countries . Camels Flaviviridae, genus Pestivirus, single-stranded linear RNA [Bunyaviridae, genus Phlebovirus (80-110 nm in diameter), 3-segmented , single-stranded negative-sense RNA genome, and single serotype [Bovine viral diarrhea virus (BVDV) and rift valley fever virus (RVFV) have the similar potential outcome in causing reproductive and respiratory manifestation in camel ,5. BVDV near RNA  that croserotype  that is Bovine viral diarrhea is one of the important viral diseases causing respiratory distress and abortion, while rift valley fever is one of the most serious zoonotic transboundary animal diseases. The World Organization for Animal Health (OIE) defined them as a communicable disease which has the potential for serious and rapid spread, irrespective of national borders. They have a serious economic impact on the animal production sector as causing losses from reproductive failures and increase the severity of secondary infections by other pathogens .In Egypt, BVD was considered as an endemic disease before 1992. BVD virus was first isolated in 1975 from calves suffering from severe enteritis , and sinThe majority of herd diagnosis for the detection of BVDV and RVFV infections is often based indirectly on an interpretation of serological tests of animal screenings. Although there are some reports on BVDV and RVFV infection and genotyping of the isolated viruses in Egypt, still there are no appropriate data on surveillance and percentage of infection because those reports are often based on selected cases. This makes it difficult to estimate the real percentage of the disease in animals .There are various methods available for diagnosing acute or persistent BVDV infections. These include immunohistochemistry, several antigens (Ags) and antibody enzyme-linked immunosorbent assays (ELISAs), reverse transcription polymerase chain reaction (RT-PCR) assays, and virus isolation . The serThe present study was designed to investigate the current epidemiological surveillance of BVDV and RVFV infections of camels originating from Sudan “smuggler” and local breed as part of our future plan for a national surveillance program throughout the country, which will aid in controlling both diseases. This investigation was accomplished using serological and molecular diagnostic techniques.This study was approved by the Research Committee of the Faculty of Veterinary Medicine, Benha University, Qualyobia, Egypt.A total number of 200 blood samples were collected from camel  and were subjected for the investigation of BVDV and RVFV infections with different age and sex. In addition, 20 buffy coat samples  were collected from febrile animals and stored at −20°C for molecular detection of BVDV and RVFV Ags under the national and international standard biosafety conditions and ethics.The clear serum was obtained using sterile Pasteur pipettes from the blood samples were left in tightly closed tubes overnight at 4°C and then centrifuged at 3000 rpm for 10 min to separate the sera and non-sera fractions. The clear serum placed in Eppendorf tubes, labeled, and stored at −70°C in the laboratory for BVDV Ag, BVDV antibodies, and IgG antibodies of RVFV using ELISA test as serological test.® compact ELISA, Ingenasa, Spain) according to the manufactures’ instruction.Antibodies detection against BVDV was performed on camel serum samples using the commercial kit, . RIFTC VER 0411 GB, ID according to the manufacturer’s instruction.Anti-RVFV nucleoprotein antibodies were detected by ID screen® Viral RNA Mini Kit  following the mini spin protocol according to the manufacturers’ instructions. About 1 μL of the obtained RNA was used as the template in a one-step RT-PCR .The viral RNA was extracted from buffy coat samples (ELISA-positive samples) by QIAampThe primers used were forward primer O1100 5’-CATGCCCTTAGTAGGACTAGC-3’ and reverse primer Ol 380R 5’-AACTCCATGTGCCATGTACAG-3’.The amplification reaction was carried out using Thermal Cycler  in the following cycles: The first step was the reverse transcription step, this step was carried out at 50°C for 30 min for one cycle, then followed by initial denaturation at 94°C for 15 min for one cycle. After that 40 cycles of amplification in three steps consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were performed, finally one cycle of final extension at 72°C for 10 min then holding at 4°C∞. The PCR products were placed on a gel plate prepared using Tris–EDTA buffer containing 1.5% agarose gel, stained by 2 μLGelRed™ stain  then electrophoresed at 100 V for 30 min, and finally visualized by ultraviolet light transilluminator (BioRAD) after using a 50 bp DNA ladder.The obtained RT-PCR products were purified by Qiaquick PCR Purification Kit (Qiagen) according to the manufacturers’ instructions. The RT-PCR products were eluted in nuclease-free water to be ready for sequencing purpose.Sequence analysis was carried out to detect the nucleotide and amino acid composition of the detected strain. It was carried out in both directions using the previously mentioned forward primer and reverse primers by 3730 DNA Analyzer, Applied Biosystems, USA. BigDye Terminator v3.1 Cycle Sequencing Kit  was used as recommended by the manufacture protocol with small modificationsMultiple sequence alignment and phylogenetic analysis were applied for sequence alignment and construction of phylogenetic tree to detect the genetic relatedness of the tested strains compared to other worldwide strains registered in the GeneBank.BVDV NS2-3 protein sequences from different localities registered in GeneBank were compared, aligned, and analyzed using BioEdit Software Program V.5.0.9, USA [The nucleotide sequence data obtained after genetic analysis in DNA Analyzer “286 nucleotide sequences were used in analysis,” and the data from others 0.9, USA .Phylogenetic analysis was carried out by constructing neighbor-joining (NJ) tree of the nucleotide sequence data using the MEGA 7 software program with the evolutionary model set to Kimura 2-parameter + Gamma. The reliability of the phylogenetic relationships was evaluated using non-parametric bootstrap analysis with 1000 replicates for NJ analysis ,24.®Viral RNA Mini Kit  following the mini spin protocol according to the manufacturers’ instructions. About 1 μL of the obtained RNA was used as the template in a one-step RT-PCR .The viral RNA was extracted from buffy coat samples (ELISA-positive samples) by QIAampThe primers used were forward primer 5’-CCTTAACCTCTAATCAAC-3’ and reverse primer 5’-TATCATGGATTACTTTCC -3’ .The amplification reaction was carried out using Thermal Cycler  in the following cycles: Reverse transcription was applied at 50°C for 30 min and a primary denaturation step was done at 95°C for 5 min, followed by 35 cycles of 94°C for 30 s, 55°C for 40 s, and 72°C for 50 s min. A final extension step was done at 72°C for 10 min. The PCR products were placed on a gel plate prepared using Tris–EDTA buffer containing 1.5% agarose gel, stained by 2 μLGelRed™ stain , then electrophoresed at 100 V for 30 min, and finally visualized by ultraviolet light transilluminator (BioRAD) after using a 50b pDNA ladder.The statistical analyses were performed by univariate analysis of variance, one-way ANOVA, and independent t-tests using SPSS program version 20 . p<0.05 was assumed for statistical significance.The results of the serological survey for BVDV and RVFV from camels are shown in The frequency of positive cases was significantly different between origin of samples and sex and age of camel for BVDV and RVFV as shown in From the febrile camel, seven buffy coat samples of BVDV-positive serological analysis and five buffy coat samples of RVFV-positive serological analysis were submitted for rapid detection by one-step RT-PCR. We found that three samples of seven were positive with RT-PCR for BVDV of camel originated from Sudan (smuggler) and successfully amplified 286 bp corresponding to the BVD genome. On the other hand, no RVFV Ag was detected in all five samples.The amplified amplicons of BVDV RT-PCR-positive samples were extracted from the gel after electrophoresis, then purified, and finally succumbed to sequential analysis. The sequence analysis was carried out using BigDye termination method in both directions using the above-mentioned forward and reverse primer, and then, the result was analyzed using 3730 DNA analyzer.BVDV NS2-3 encoding gene (from nucleotides 105 to 390) and amino acid sequences (from 35 amino acids to 130) of the full-length BVD genome were aligned and analyzed.After direct sequencing, 286-nucleotide sequences corresponding to partial The obtained data revealed 100% nucleotide homology with Sudan strain 2015 except only one (missense point mutation) by substitution of A to T at position 345 that changed the coded amino acids from T (Threonine) to S (Serine) at residue 115.Phylogenetic analysis was carried out to detect the genetic relatedness of the isolated strain in the current study with other strains worldwide expressed as accession numbers .Phylogenetic analysis was carried out by constructing NJ tree of the nucleotide sequence data using the MEGA 7 software program with the evolutionary model set to Kimura 2-parameter + Gamma. The reliability of the phylogenetic relationships was evaluated using non-parametric bootstrap analysis with 1000 replicates for NJ analysis.The results of the phylogenetic analysis indicated that the isolated strain in this study was clustered with Sudan strain, proving the same source of infection.Surveillance is an important defense in our country’s resilience against animal diseases. It is the continuous monitoring of the occurrence of disease within a population through the collection, collation, analysis, and dissemination of disease-related data. This information is used as a guide to the management of diseases within populations through planning and evaluating strategy measurement to prevent and control disease. Animal surveillance has and will continue to be an important component of the veterinary infrastructure supporting livestock industries, food production systems, and rural economies around the world.Camels may play a role in the persistence and transmission of many diseases through direct contact with domestic animals, such as sheep and goats, and to a lesser extent with cattle in Egypt, especially BVDV and RVFV diseases ,27.et al. [et al. [In a serological survey, the incidence of BVDV antibodies and Ag in 200 camels tested was 47.5% for antibodies, and 31.6% for Ag in camel originated from Sudan, but in local breed camels, it was 11.2% for BVDV antibodies and 7.5% for BVDV Ag. These findings indicated that there is a failure in the quarantine measures which are applying for live camels exporting from Sudan. The previous findings disagree with the findings of Saidi et al.  who founet al.  who reco [et al.  detectedet al. [On the other hand, the incidence of RVFV IgG antibodies was 16.7%, and 8.7% in the examined camels originated from Sudan and local breed, respectively. There are previous studies in Egyptian camels during the present interepidemic period which was conducted in 2009/2010 with 10 camel sera from an abattoir and in 2012 encompassing 100 camels from Qualyobia Governorate. Likewise, no further information about the origin of the animals was given in these reports. Both studies failed to detect antibodies in Egyptian camels. Only 10% of imported animals are tested against RVFV antibodies directly after the import as a routine disease control . The riset al.  who founThe incidence of BVDV antibodies, BVDV Ag, and RVFV IgG antibodies was the highest in the age group of ≥9 years. Furthermore, there is a highly significant correlation between the effect of age and incidence of BVDV antibodies, BVDV Ag, and RVFV IgG antibodies in the examined camel serum samples. This might indicate that older animals have a higher exposure to infection and contact could have occurred some years ago ,34.et al. [The proportion of the positive camels for BVDV antibodies, Ag, and RVFV IgG antibodies were the highest in male than female. There was a statistical difference between the sexes with BVDV antibodies, Ag, and RVFV IgG antibodies in the examined camel serum samples. This may be due to the fact that male camels travel from one place to another place to provide transportation service more than female camels so that they have a higher probability of acquiring an infection. Frequent travel could also compromise their immune response to infection due to the stress of fatigue. These findings disagreed with the results of Raoofi et al.  who founThe results indicated that camels might represent an important source for BVDV infection in all ruminants, including cattle, sheep, and goats breed in mixed herds since they had higher BVDV prevalence rates. Therefore, the prevention and control measures for BVDV spreading and persistence the camel should house in camel populations only to limit the spread of BVDV infection to ruminant populations . The resCamels consider as the contagious population in Egypt so play an important role in the transmission of many diseases, especially BVDV and RVFV, throughout traveling of camel from country to another country. Based on these study findings, continuous disease surveillance of camels for BVDV and RVFV is indicated, which is a good tool for eradication, control of such infectious diseases, and measuring RVFV circulating activity in non-vaccinated camels. Finally, the difference in the prevalence among countries may due to numbers of animal used, occurrence of vector, and previous factors such as vaccination and quarantine measures.Camels act as risk animals for the introduction of many infectious diseases from Sudan to Egypt, especially transboundary animal diseases, so strict quarantine measures should be taken during importation of live animals from Sudan to prevent the spread of such diseases. The control of such diseases was submitted to main points as routine annual surveillance, vaccination of animals, and vector control.HEKE, HKA, and MAM designed the concept for this research and scientific paper. HEKE has conducted the maintenance of camels used in the experiment, collecting samples, and compiling the resource materials. HKA provided technical supports and made serological examination of serum samples. HEKE analyzed the serological data. MAM conducted the molecular detection of BVDV and RVFV in buffy coat sample and sequencing of BVDV. All authors participated in the manuscript’s draft and revision. All authors have read and approved the final manuscript.


In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves , the technical environment , or the human microbiome . In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable  contains supplementary material, which is available to authorized users. The long-term storage of biomaterials (biobanking) is a precondition for modern life sciences, enabling follow-up scientific investigations, medical diagnostics, biotechnological applications, and the conservation of genetic resources and diversity Overmann . To guarCryopreservation constitutes a key component of contemporary biobanking. Specific cryopreservation protocols have been established for different organisms and cell types. Living biological material may be prepared for cryopreservation under both, sterile or unsterile conditions. As a result, the biological materials themselves as well as the storage facilities may contain additional, accompanying organisms. For instance, plant material and human or animal cell material may be colonized by viral or bacterial pathogens  were used for DNA extraction. A total volume of 10 ml of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid  buffer  was added and the tubes were incubated at room temperature on a horizontal shaker to resuspend the residuals for 15 min. Samples were filtered through a 0.1 μm pore-size polycarbonate filter and stored at −20 °C for DNA extraction. For microscopic counting, the residuals from four pooled 50 ml  LN subsamples were resuspended in 5 ml HEPES by shaking for 15 min and then fixed with glutaraldehyde . The ice samples were thawed and HEPES buffer was added up to 10 ml in samples containing less than 10 ml thawed ice. An aliquot of 1.8 ml of each sample was fixed with glutaraldehyde for microscopic counting and the remaining suspension filtered through a 0.1 μm pore-size polycarbonate filter.Three types of negative controls were included. Firstly, empty Falcon tubes provided along with the samples by each participant served as negative controls for the contamination of laboratory equipment, they were filled with 25 ml HEPES (Negative Control = NC eq). Secondly, reference samples processed at DSMZ consisting of 550 ml of the LN were filled into a sterilized Dewar, 0.1 μm Isopore™ polycarbonate filters  added, and the LN evaporated (NC ref). Thirdly, two of the 0.1 μm polycarbonate filter were treated by filtering 25 ml HEPES (NC HEPES). All controls were processed in parallel and in the same way as the samples.a autofluorescence as indicator of the presence of algae or plant cells using a Nikon  Ti microscope at an excitation wavelength of 425 nm and an emission wavelength of 607 nm and Nikon software NIS-Elements AR 4.13.01.For fluorescence microscopy, fixed cells were collected onto polycarbonate filters , the filters were stained with 50 μl SYBR Green I  and mounted in a drop of immersion oil on a glass slide. The samples were analyzed using a Zeiss  Axio Imager.M2 microscope at excitation/emission wavelengths of 470/525 nm, and the Axio vision software Rel. 4.8.2. Twenty microscopic fields were counted in triplicate for each sample. Conspicuous structures were analyzed further for the presence of chlorophyll DNA was extracted from the filters using the DNA Micro Kit  according to the protocol of the manufacturer. Filters were cut into strips and incubated with lysozyme  at 37 °C on a shaker (800 rpm) for 1 h. In the second lysis step, 20 μl proteinase K  was added and the samples were incubated at 56 °C over night. In the final step of the protocol, DNA was eluted in 20 μl PCR-clean water .Mycoplasma was detected using a previously established PCR-based detection method  transposable elements Line1, and fungal ITS region were PCR amplified using the respective primer sets 8F-1492R, Line1 and ITS1F-ITS4 at a final concentration of 0.2 pmol per μl Table . The PCR2 (0.5 mM), DMSO (3.0%), and PCR-clean water. Between 1 and 20 ng, DNA template was used. The PCR product (60 μl) was cleaned up using DNA Clean & Concentrator™-5  eluting the product in 30 μl water. After adding 0.1X TE  to a final volume of 50 μl, the amplicon was processed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®  according to the protocol of the manufacturer. Amplicons were prepared for adapter ligation using the NEBNext End Prep enzyme mix, and the 25-fold diluted adapter was ligated in a subsequent step. Adapter-ligated fragments were cleaned up without size selection using Agencourt AMPure XP Beads . Then, the adapter-ligated DNA was enriched by 13 PCR cycles of using NEBNext® Multiplex Oligos for Illumina® . The size distribution of the purified PCR product (AMPure XP Beads) was checked on an Agilent Bioanalyzer . Adapter dimers of the combined library pool (~ 10 ng PCR product per sample) were removed by gel purification  using the NucleoSpin® Gel and PCR Clean-up Kit  and amplification products were sequenced on a HiSeq 2500 Ultra-High-Throughput Sequencing System  as described recently , Qiagen Phusion polymerase  and GC-buffer with the addition of dNTPs (0.2 mM), BSA (0.8 mg per ml), MgCl https://qiime2.org/) creating a Feature Table. Default settings were used unless otherwise noted. The forward and reverse reads were joined, chimera-filtered and clustered . The reads were then compared against SILVA 132 SSURef Nr99 with an initial identity cutoff of 97% with vsearch 2.7  using plugins implemented in Quantitative Insights into Microbial Ecology  using the adonis2 function of the vegan package in R , Constrained Analysis of Principal Coordinates , and relative abundances of the bacterial communities were calculated on genus level removing sequence variants with abundances of less than 5 reads per sample phyloseq package  and of the negative controls  and gene copies  could be detected in over 90% of the samples from all institutes Fig.  with excp < 0.01, excluding seldomly opened tanks).The bacterial species richness determined on the genus level for individual LN storage tank samples typically stayed below 300 sequence variants. Higher values were only determined in five individual samples Fig. a. Using Notably, species richness and diversity were low in the debris samples (< 50 species) despite the relatively high cell numbers in those samples. Samples storing mixed material showed significantly higher species richness and diversity than samples storing animal material Table . The envp < 0.001). Strengthening these results, the three consecutive samplings of the same LN storage tank did not yield significant differences in the bacterial community composition. Bacterial communities detected in the ice phase and in debris were more variable than those detected in the LN phase or the negative controls  and the variability in the 20 most abundant genera could be assigned to certain conditions and samples. The opening frequency seemed to influence the bacterial community of LN storage tanks in institute C, separating LN tanks No. 10 and 11 from No. 8 and 9. Interestingly, samples that were tested positive for fungi and Mycoplasma-DNA with PCR also harbored a specific bacterial community or particular bacterial taxa.For this purpose, the ordination was constrained by the environmental variables. About 48% of the variability was explained by the first two principle coordinates , while in sample No. 18 that clustered in the same group of bacterial communities as samples No. 12 and 36 , Bradyrhizobium (Alphaproteobacteria), Pseudomonas saccharophila (Gammaproteobacteria), Sphingomonas (Alphaproteobacteria), and Bacillus (Bacilli) are typical representatives of the most abundant phyla in the LN storage tanks , Staphylococcus epidermidis, and Staphylococcus pasteuri . Most of the sequences of sample No. 20 were affiliated to Pedobacter glacialis  from a single LN storage tank of institute H storing microorganisms  that has been proposed for cleanrooms , especially for sensitive applications like transplants, because a Mycoplasma infestation renders the biological resource useless in these cases and results in a financial burden. Nevertheless, based on the results of this study, the rather rare occurrence of fungi and eukaryotic cells is expected to have little effect on biobanking. Chloroplasts occur more frequently than the other specific groups investigated, especially when mixed samples are stored. Yet the accompanying plant materials are not considered as a threat for the stored bioresources since they typically are not pathogenic. The exceptional high amount in single samples may be caused by material released due to failure of container seals.The genus In summary, the microbial cell concentration was clearly depending on the institutes and their specific storage conditions. We therefore conclude that individual measures taken by biobanking institutes exert a profound effect on the quality of the cryostored biomaterials.The bacterial species richness for LN storage tank samples is much lower compared to species richness of soils  frequent occurrence in the environment, (2) presence on/in the operating personnel, (3) their specific physiological adaptations, or (4) the introduction into LN tanks in association with the stored biomaterials.Methylobacterium are able to form biofilms that tolerate disinfecting agents, high temperatures, or low water availability and as a result occur widespread in man-made environment such as industrial storage tanks . They might originate from the stored plant material, which was stored in non-hermetically sealed tubes in the respective LN tank. Devices which are not hermetically sealed may allow the exchange between the cryopreserved materials and the surroundings  or plants-associated species , Bradyrhizobium (Alphaproteobacteria), Pseudomonas saccharophila (Gammaproteobacteria), Sphingomonas (Alphaproteobacteria), and Bacillus (Bacilli), which are typical representatives of the most abundant phyla in the LN storage tanks, have also been isolated from ultrapure water in industrial systems , Staphylococcus epidermidis, and Staphylococcus pasteuri are part of the commensal mammal microbiota of skin, hair, and nail. Staphylococcus pasteuri has even been isolated from clean rooms , Bacillus, Propionibacterium, and Staphylococcus in LN tanks  were also found in the amplicon sequences. Since Elizabethkingia and Empedobacter include some pathogenic species


Seagrass meadows play a key ecological role as nursery and feeding grounds for multiple fish species. Underwater Visual Census (UVC) has been historically used as the non-extractive method to characterize seagrass fish communities, however, less intrusive methodologies such as Remote Underwater Video (RUV) are gaining interest and could be particularly useful for seagrass habitats, where juvenile fish camouflage among the vegetation and could easily hide or flee from divers. Here we compared the performance of UVC and RUV methodologies in assessing the fish communities of two seagrass meadows with low and high canopy density. We found that RUV detected more species and fish individuals than UVC, particularly on the habitat with higher seagrass density, which sheltered more juveniles, especially herbivorous, and adult piscivorous of commercial importance, evidencing significant differences in energy flow from macrophytes to predators between seagrass habitats, and also differences in the ecosystem services they can provide. Considering the ongoing worldwide degradation of seagrass ecosystems, our results strongly suggest that fish surveys using RUV in ecologic and fisheries programs would render more accurate information and would be more adequate to inform the conservation planning of seagrass meadows around the world. Seagrass attain some of the highest rates of primary production in aquatic environments6, helping to maintain good water quality by absorbing excess of nutrients and by trapping and stabilizing sediments9. Iconic organisms such as marine turtles and sirenians (manatees and dugongs) are highly dependent on seagrass habitats10. Unfortunately, the importance of seagrass meadows has been particularly overlooked in tropical environments, where the attention is focused on the colorful coral reef communities11. Seagrass ecosystems provide many goods and services to the human societies14. Among these, their fundamental role as nursery and feeding grounds for fish of ecological and commercial importance is highly valuable20. However, seagrass meadows are severely affected by multiple anthropogenic disturbances22, including heavy exploitation of their fishery resources23.Seagrass meadows provide shelter and food resources that sustain high levels of marine biodiversity in complex trophic chains interconnected with adjacent ecosystems such as mangroves and coral reefs25. Indiscriminate extractive methodologies using different types of nets, such as throw traps, drop samplers, seine and trawling nets , as it is a low-cost technique, which allows fast data gathering , consists on a platform holding a battery-operated video camera inside a waterproof housing. Nowadays, action cameras offer a perfect balance between price, image quality, operability and inconspicuousness31. This approach allows recording less disturbed fish communities, reducing the bias in the assessment of community structure. Moreover, RUV has fewer limitations of time and depth, facilitating replication at wide spatial scales. The number of replicates obtained at the same time will depend on the number of video units in possession32. Following standardized procedures, RUVs can be used on different aquatic habitats25, minimizing the comparative problems presented by other approaches33. In addition, videos are permanent records, allowing future comparative analyses and can be used for campaigns of public awareness30.The development of video-sampling methodologies to study marine communities dates back from the 50 s, although it is not until recently that its use has spiked30. The literature review we performed showed that only 13 studies have used video samplings to assess seagrass fish communities  species diversity and composition, (ii) abundance of different fish life-stages; and (iii) trophic guilds of seagrass fish communities. We selected two types of tropical seagrass meadows characterized by contrasting canopy morphology and density of the main habitat-builder Thalassia testudinum: Backreef, characterized by shorter leaves, lower Leaf Area Index (LAI) and above-ground seagrass biomass; and Lagoon, having longer leaves, larger LAI and higher above-ground biomass. Both habitats were previously described35.The application of video-based methodologies is almost restricted to Oceania, USA and Europe, with marine biodiversity hotspots such as the Coral Triangle and the Caribbean Sea lagging behind1, 24 = 31.408, P = 0.0001; Lagoon: pseudo-F1, 26 = 38.707, P = 0.0001) and species .Both methods found higher species diversity in the Lagoon but RUV detected higher numbers of species and families on both habitats. We identified a total of 63 fish species belonging to 26 families, with RUV recording 48 species of 25 families and UVC registering 31 species of 17 families in total. RUV found 38 species (22 families) at the Lagoon and 34 species (18 families) at the Backreef, with 24 species (15 families) in common, whereas UVC registered 19 species (14 families) in the Lagoon and 16 species (9 families) at the Backreef, with only 4 species (6 families) found in both meadows. There were no significant differences in average species and family richness between habitats for both methods. However, differences between methods were significant in each habitat  than UVC (21) at the Lagoon after 14 sampling units and was predicted to reach 30 ± 9.8 species if sampling effort was increased to 24 RUVs, close to the prediction for UVC: 30 ± 11.7 Fig. . HoweverP = 0.2376). However, RUV recorded higher abundances than UVC . For juveniles UVC found slightly higher abundances at the Backreef . In contrast, RUV’s data showed that the Lagoon sheltered higher amounts of juvenile fish .The fish communities characterized by both methods also differed in fish abundance. RUV registered significantly more individuals of all life stages than UVC in both seagrass meadows with the exception of juveniles at the Backreef Fig. ; Table 1P = 0.001) and RUV , however, each methodology registered different species assemblages . Species of the families Acanthuridae, Labridae, Chaetodontidae, Pomacentridae and Haemulidae were preferentially detected by UVC, whereas species of the families Sphyraenidae, Gerreidae, Tetraodontidae, Balistidae, Ostraciidae, Carangidae, Mullidae, Lutjanidae, Scaridae and Monacanthidae were predominantly detected by RUV , seconded by acanthurids (1.69 ± 0.61), scarids (0.92 ± 0.35) and haemulids (0.53 ± 0.53), while among the adult fish registered the most abundant were labrids as well (0.46 ± 0.38). At the Lagoon, juvenile fish were mostly lutjanids (7.07 ± 6.99), labrids (2.64 ± 0.84), haemulids (2.21 ± 1.30), and scarids (1.57 ± 0.56), while the most abundant adult fish were carangids (1.57 ± 1.57) and labrids (1 ± 0.85) Fig. . In comp1, 25 = 18.64, P = 0.0004), while at the Lagoon benthopelagic carnivores were more abundant (9.42 ± 7.20) than benthic carnivores (3.07 ± 0.77) and herbivores . Similarly to UVC, the surveys of RUV showed that benthic carnivores were the most abundant at the Backreef , however, the abundances of benthic herbivores (11.76 ± 3.81) and benthopelagic carnivores (5.38 ± 1.55) were higher. At the Lagoon, the pattern was different, with benthic herbivores (24.35 ± 8.10) and benthopelagic carnivores (25.5 ± 13.63) having higher abundances than benthic carnivores   and schools of Caribbean squids .The patterns previously described for UVC were mainly based on juvenile individuals, since few adults were detected Fig. . In conti.e. MaxN) is a conservative measure designed to avoid double counting, and thus represents the minimum abundance per species. Accordingly, it is still likely that we underestimated real abundances of fish populations with high densities37. Our analysis indicates that 55 minutes of recording with the RUV method approached the asymptote of the species richness curves much faster than lower recording times or UVC transects. This agrees with the literature in that recording for more than 30 minutes may be more cost-effective to assess fish diversity in coral reefs38 and seagrass meadows39. The ability of RUV to carry out higher sampling effort (as time) per location, combined with the absence of the disturbance associated with the presence of divers40, may explain its higher effectiveness for describing fish communities.The comparison performed here highlighted significant differences between Underwater Visual Census (UVC) and Remote Underwater Video (RUV) in their effectiveness to characterize fish communities of tropical seagrass meadows at worldwide scale. RUV recorded higher species diversity and abundance of fish individuals of all life-stages and trophic groups than UVC, despite the fact that abundance data recorded by RUV  found 28 species after 48 transects of 20 meters42. Although a direct comparison of our results with these previous studies is not possible due to spatial and temporal mismatches43, the values show a good approximation to the sampling effort predictions of our species accumulation curves, where UVC would require more than double the sample units to approximate the species richness found by RUVs of 55 minutes. These differences could be even higher, as we carried out UVC transects before setting up the RUV units, which might have caused some fish to flee. We considered five minutes of buffer between techniques, which is a common time frame used in comparative studies40. However, this might have not completely off-set the “diver-effect” in the RUV results27.Past surveys on seagrass habitats on the northern Mexican Caribbean (Cancun and Puerto Morelos) registered 62 species of fish after 36 UVC transects of 50 meters44. However, both techniques have been found to differ on species composition, depending on the habitat and behavior of each species. The higher the topographic complexity of the reef habitat, the more effective UVC could be, since fish can avoid being recorded in videos by sheltering on caves, cracks or overhangs47. Species with wary behavior that tend to flee from divers have higher probability to be recorded by RUV and bold species such as site-attached fish, can be easily detected by UVC, while mobile conspicuous species, such as labrids, present similar likelihood to be detected by both methodologies44. Our results partially agreed with these past observations. However, the seagrass environment seemed to dwarf some of the benefits traditionally attributed to UVC, as we found that RUV characterized better the fish communities, particularly the habitat with higher canopy density. In agreement with previous studies UVC registered seven site-attached species of haemulids, chaetodontids, pomacentrids and sciaenids that were not recorded by RUV  on RUV systems58. In our study we didn’t use bait, but still found that RUV recorded more adult individuals than UVC, particularly more carnivores valued in the fishing industry. Indeed, RUV recorded higher number of species and individuals in adult stage, describing more accurately trophic interactions in the ecosystem, as the few adult organisms registered by UVC surveys depicted incomplete trophic patterns. The use of bait in RUV sampling reefs has been an effective way to attract more individuals and species to the video cameras59. However, its use in seagrass meadows may not be so positive, as we observed in the videos that juvenile fish rushed for shelter when carnivorous fish such as Caranx ruber were swimming close by. As multiple predators may remain permanently around the camera in the presence of bait, many juvenile fish would be scared away, impacting the estimations of abundance and species richness.Fish surveys using video techniques in reef systems have registered more adult predators than diver-based methods, this could be attributed to the addition of bait  could explain the higher number of juvenile herbivorous64. At the same time, the higher abundance of juveniles may attract piscivorous species from nearby habitats65. However, the protection provided by the more dense seagrass canopy may reduce mortality rates, maintaining high abundances of juveniles at the Lagoon66. In contrast, benthic carnivores were dominant in the Backreef, although at the same abundance than in the Lagoon, suggesting that they could feed equally on both habitats. Ostracids were observed roaming and feeding in both habitats, but we just saw dasyatids digging and ingesting sediments at the Backreef. This suggests that less dense canopies may facilitate access to the sediment, making easier to dig out invertebrates, while denser canopies may provide more food among and/or on the leaves. The patterns of abundance, species richness and trophic groups documented here are likely to change in space and time67 associated with diurnal changes68 and seasonal fluctuations in water temperature and irradiance. These factors determine the reproduction timing of many fish species, as well as changes in canopy structure and in the primary production of the seagrass habitat, which in turn affects its shelter capacity and the amount of food resources available69.The trophic patterns documented seem to be related to the availability of food resources in each seagrass habitat, as previously reported37. Sizes can be estimated by using calibration scales46, using parallel lasers70 or using allometric relationships, such as eye to head-height proportions71. The limitation of all these approaches is that not all individuals sighted can be sized. The mean error using calibration scales was estimated as 14.3 ± 2 mm72 and has been applied successfully in large scale ecological studies74. In contrast, fish sizes are routinely estimated in almost all the fish while performing UVC. However the accuracy of these estimations are highly variable and dependent on the skills of the diver, the environmental conditions and the size of the fish. UVC accuracy errors have been estimated to be −20.1 ± 0.6 mm under controlled pool conditions75, while in the natural environment divers tended to underestimate the length of small fish (175 mm) by 35 mm and overestimate big fish (400 mm) by 40 mm76. Stereo-video systems have been developed to overcome these biases and can successfully measure length with minimal error (0.2 ± 0.4 mm)72. The drawback of this technology is that the equipment for sampling, storage information and the required software for video analyses increase the costs of the survey program considerably. However, as these technologies advance and their use increases, their accessibility is also enhanced77. An example can be found in Cuba, where researchers have been able to operate stereo-video technologies since 201178.An additional point to be considered is the capacity of RUV methodologies to determine fish sizes, which can be used to calculate biomass through known length-weight relationshipsAs the degradation of seagrass meadows progresses around the world, the need to plan and support conservation actions becomes urgent, therefore accurate environmental and biological information is needed. Our study demonstrates that Remote Underwater Video (RUV) is a more effective tool than Underwater Visual Census (UVC) for describing the fish communities of seagrass meadows. The finding suggests that past descriptions relying on UVC surveys could have underestimated the abundance and species diversity of seagrass habitats, especially under limited sampling efforts. The fact that our analysis was performed in a tropical environment with high levels of fish diversity supports the utility of video technologies to sample in other regions with lower diversity such as temperate habitats. The use of a common methodology in different parts of the world is fundamental for comparative studies. For these worldwide comparative purposes, the use of techniques that carry less bias is particularly recommendable. The results obtained using RUV surveys highlighted the importance of the density of the seagrass canopy for structuring and maintaining the fish communities of marine coastal ecosystems. By extension, these results are also relevant to preserve those communities with particular value for recreational and commercial fisheries. Our results indicate that changes in seagrass abundance, but in particular the loss of seagrass cover will severely impact fish abundance and biodiversity, in addition to other losses in marine resources and ecosystem services. This study therefore strongly recommends RUV methods for fish surveys within ecological, conservation and fisheries monitoring programs carried out on seagrass meadows. We especially encourage its use in tropical regions, where the application of video-technologies is still lagging behind, despite the higher biodiversity and inter-connectivity of seagrass meadows with other important ecosystems such as coral reefs and mangroves.79. The morphotype of the dominant seagrass Thalassia testudinum changes significantly within the lagoon (see Enríquez and Pantoja-Reyes35). In the middle, where the sandy sediment is deeper, seagrass above-ground biomass and canopy height are higher. However, as the seagrass approaches the back-reef hard substrate is more present and the sediment becomes thinner. Seagrass meadows nearby the back-reef have reduced above-ground biomass and the canopy becomes less dense and with shorter leaves35.The study was carried out in the Mexican Caribbean within Puerto Morelos National Park, located at the northernmost section of the Mesoamerican Barrier Reef. This marine system presents fringing reefs located about 1 to 2 km away from the coast. The reefs buffer the energy of the waves and allow the development of extensive inshore reef lagoons, 4–5 m of maximum depth, where seagrass meadows developi.e. same number of sampling units than UVC). To enhance the detection of juvenile and cryptic species our video camera was set horizontally but slightly facing downwards so that the substrate covered ¾ and the water column ¼ of the video frame. No bait was used (e.g. BRUV), as we were interested on the effect of habitat structure to the fish community and bait releases produce an odor plume that can extend for hundreds of meters, attracting carnivorous fish from adjacent areas and disrupting the original community at the sampling point. Videos were recorded at a resolution of 2.7 K with medium field of view and 60 frames per second. We retrieved the video cameras after 1 hour, a common recording time  and in three main groups of trophic level and residency behavior combined for simplicity: benthic herbivores (diet based on macrophytes), benthic carnivores  and benthopelagic carnivores . Fish species identifications and categorization of life-stage, trophic group, habitat associations and commercial importance were based on the field guides81 and information available on FishBase82.We characterized here the fish communities inhabiting seagrass habitats with low (Backreef) and high canopy density (Lagoon) at three sites  located in the northern Mesoamerican Barrier Reef System (from Cancun to Puerto Morelos). In each sampling point we performed underwater visual census (UVC) along 50 × 3 m transects (7 ± 1.2 min), a common sampling area in seagrass surveys  and Analysis of Similarity (ANOSIM) to test for statistical significance. PERMANOVA Tests were performed with the software PRIMER 6 & PERMANOVA + (PRIMER-E Ltd) and metaMDS and anosim for ordination analyses were done with the program R (The R project) using the package Vegan83. Abundance data was square root transformed to reduce the effect of schooling species. All tests were based on 9999 permutations and resemblance measures from bray curtis distances.Statistical differences in families and species richness and total abundance  by different life-stages between methods (UVC and RUV) and seagrass habitats (Backreef and Lagoon) were statistically tested with two-way Permutational Multivariate Analysis of Variance (PERMANOVA) with Method and Habitat as fixed factors and Site as random factor, followed by pairwise comparisons84.Species accumulation curves were generated for each habitat and sampling methodology, considering different recording length-times for RUV. For this, we used the package iNEXT (iNterpolation and EXTrapolation) of the software R, which uses rarefaction to calculate species accumulation and sample completeness curves based on the species presence/absence data and predict new species detection with future sampling effort; it also generates confidence intervals for the curves (±95%) by bootstrappingAll graphics in the manuscript were generated with the package ggplot of the software R (The R project). Panel labels and photographs were added using the graphics software GIMP 2. Photographs in Fig. Supplementary tables


Both mandibular condylar hyperplasia and condylar osteochondroma can lead to maxillofacial skeletal asymmetry and malocclusion, although they exhibit different biological behavior. This study attempted to compare the histological features of mandibular condylar hyperplasia and condylar osteochondroma using hematoxylin-and-eosin (H&E) staining, and immunohistochemistry staining of PCNA and EXT1 with quantitative analysis method.P = 0.018). The percentage of bone formation in condylar osteochondroma was larger than was found in condylar hyperplasia (P = 0.04). Immunohistochemical staining showed that PCNA was mainly located in the undifferentiated mesenchymal layer and the hypertrophic cartilage layer, and there were more PCNA positive cells in the condylar osteochondroma (P = 0.007). EXT1 was mainly expressed in the cartilage layer, and there was also a higher positive rate of EXT1 in condylar osteochondroma (P = 0.0366). The thicker cartilage cap, higher bone formation rate and higher PCNA positive rate indicated a higher rate of proliferative activity in condylar osteochondroma. The more significant positive rate of EXT1 in condylar osteochondroma implied differential biological characteristic as compared to condylar hyperplasia.The H&E staining showed that condylar hyperplasia and condylar osteochondroma could be divided into four histological types and exhibited features of different endochondral ossification stages. There was evidence of a thicker cartilage cap in condylar osteochondroma as compared condylar hyperplasia (These features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma. Osteochondroma is described as osteocartilaginous exostosis . It is cNow differential diagnosis of these two mandibular diseases tends to depend on non-invasive diagnostic examination, including X-ray, CT and MRI. However, these methods present with inherent limitations, and the cell behavior based on pathological information is still considered the definitive choice for diagnosis. Furthermore, studies of the pathogenesis and molecular biology of mandible condylar hyperplasia and condylar osteochondroma currently remain at an initial stage of investigation, and the qualitative H&E staining results showed no characterized cell behavior between these two diseases .Both diseases are characterized by excessive growth and enlargement of the mandibular condyle. Therefore, cell proliferation is a key evaluation marker, and a quantitative and specific staining method is necessary to effectively make a differential diagnosis of mandible condylar hyperplasia and condylar osteochondroma. Besides, PCNA (Proliferating Cell Nuclear Antigen) is a nuclear protein that is expressed in the G1-M phases of the cell cycle, but is maximally expressed in the late G1-S phase . PCNA isWhile the majority of osteochondromas present as solitary  lesions , 14, appIn this current study, we attempted to quantitatively describe the histological and molecular features of mandibular condylar hyperplasia and condylar osteochondroma. Subsequently, the histological and molecular difference between both diseases was also described and discussed.P = 0.448 < 0.05).7The diagnosis of condylar osteochondroma and condylar hyperplasia were made by experienced maxillocraniofacial surgeons, radiologists and pathologists, and diagnoses were based on the clinical symptoms, CT scanning characteristics and H&E staining Fig. . Thus, tBoth condylar hyperplasia and condylar osteochondroma showed a cartilage cap that covered the surface of the condyle. The cartilage cap was divided into four layers: the fibrous layer, undifferentiated mesenchyme layer, cartilage layer including pre-hypertrophic and hypertrophic chondrocytes and the calcified cartilage layer Fig. . The con(1) Type I Fig. a and b: (2) Type II Fig. C and D: 3) Type III  Type IV Fig. g: The unp = 0.9928) and the condylar osteochondroma group   and the chondrocyte layer  in condylar osteochondroma when compared with condylar hyperplasia. The percentage of bone formation in condylar osteochondroma was larger than found in condylar hyperplasia . Whereas the thickness of the fibrous layer, undifferentiated mesenchymal cell layer, the number of cartilage islands and the depth of infiltration of the cartilage islands were not significantly different between groups  found in the cartilaginous cap, which also involved the EXT gene location. Additionally, the loss of function or mutation of EXT1 is crucial in the pathogenesis of solitary as well as hereditary osteochondromas . The EXTThe positive rate of EXT1 expression in the condylar osteochondroma group was significantly higher than was found in condylar hyperplasia. EXT1 expression was concentrated on the cartilage layer. In addition, we can infer that over-expression of EXT1 may cause a disorder of endochondral ossification signaling cascades, leading to osteochondroma. Moreover, the negative expression of EXT1 in an all burn-out type IV specimen of condyle hyperplasia was shown to give rise to the relative relationship between EXT1 expression and cartilage formation in condylar osteochondroma.In summary, our semi-quantitative method for H&E and immunohistochemical staining showed that there was a thicker cartilage cap, a higher bone formation rate and higher PCNA positivity in condylar osteochondroma when compared to condylar hyperplasia, which indicated a higher proliferative activity of condylar osteochondroma. In addition, a higher EXT1 positive rate in condylar osteochondroma implied different biological characteristics in condylar osteochondroma when compared to condylar hyperplasia. These features might be useful in histopathologically distinguishing condylar hyperplasia and osteochondroma and in providing the basis for exploring the mechanism of condylar osteochondroma. However, its sensitivity and accuracy in clinical applications requires further study with a larger sampling set.This study was carried out at Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine. All patients were informed of the study purpose and gave consent. Eighteen cases with typical condylar osteochondroma, and 15 cases with typical condylar hyperplasia were treated in the Department of Oral and Craniomaxillofacial Surgery through 2005–2014. All cases were diagnosed based on the sequence including clinical features, representative computed tomography (CT) scan characteristics, single photon emission computed tomography (SPECT), and histopathological features  Clinical examination showed notable increases in ramous height and condyle neck height of the affected side that led to a rotated facial appearance and a canting occlusal plane. The prominence of the chin deviated to the contralateral side. In addition, temporomandibular disorder was detected in some cases.(2) CT scans showed morphological enlargement of the condyle, and elongation and thickening of the condylar neck, presenting as an enlarged and smoothed condyle. Compared with the contralateral side, the uneven ossification was more significant and the trabeculae was larger with lower CT value detected. In addition, the characteristic cartilaginous cap was not seen.(3) All cases with condylar hyperplasia were in the active phase, which was proven by follow-up visits for at least one year, with an SPECT value greater than 0.1.(4) Post-surgery histopathological examiniation gave a diagnosis of Condylar hyperplasia.(1) Clinical examination showed facial asymmetry, hypomobility, deviation of the mouth opening and malocclusion. Occlusion plane canting was also measured. Some patients showed stable occlusion when assessing progress over a prolonged period of time. In addition, temporomandibular pain, noise and pre-auricular swelling was observed in some cases.(2) CT scans showed cartilage cap covering the condylar surface and continuity of the cortex and trabeculae. The trabeculae was found to have an uneven ossification. The morphology of the condyle had clearly changed and was uneven in some cases, with lobulated surface or the formation of a pedunculated mass. The affected side of the TMJ joint surface of the temporal bone was reconstructed due to tumor compression, and the joint space was smaller than the contralateral side.(3) The SPECT value of all cases with condylar ostechondroma exceeded a value of 0.1.(4) Histopathological examination gave a diagnosis of condylar osteochondroma.Furthermore, the surgical procedures by low condylectomy and orthognathic surgery spontaneously considering facial outcome and occlusion , 7. CondThe paraffin sections were derived from the resected condyle specimens mentioned above. The sections were dehydrated and embedded in paraffin following routine methods: the specimens were fixed in 4% paraformaldehyde for 24 h at 4 °C followed by decalcification with a decalcifying solution. The samples were then dehydrated in serially graded ethanol solutions, defatted in methanol and embedded in paraffin. The condyle sections were sagittally sectioned at a thickness of 5um, and deparaffinized in xylene, rehydrated in descending concentrations of alcohol, and stained with hematoxylin and eosin (H&E).Immunohistochemistry was carried out by standard procedures. The sections had paraffin removed, which were then immersed in distilled water following routine methods. The sections were immersed in 1 mM pH 8.0 ethylene diamine tetraacetic acid  solution and then heating in a water-bath for 25 min. Next, paraffin sections were rinsed three times for 3 min each in PBS pH 7.4 at room temperature. The sections were then incubated with the primary antibody, anti-EXT1  and anti-PCNA  at 4 °C overnight in a humidified chamber. After washing in PBS, the appropriate biotin-labeled secondary antibody was applied to the specimens. After rinsing three times for 3 min each in PBS pH 7.4, sections were exposed to DAB detection solution , following which the slides were treated in alcohol and xylene and then mounted with neutral balsam.Using the smallest scale of the 0.01 mm type, and the C1 eyepiece micrometer under × 200 magnification, we selected five fields of the thickest cartilage cap area of the H&E stained sections derived from both condylar hyperplasia and condylar osteochondroma, and then measured the thicknesses of the fibrous layer, the undifferentiated mesenchymal layer, the cartilage cell layer, and the depth of average infiltration. Then the numbers of cartilage islands were calculated respectively. We also took three images of bone tissue in the thickest cartilage cap area under × 50 magnification, and used image-j2x Image processing software to process the images and calculate the percentage of the osteogenic area.P < 0.05 was statistically significant by using the SPSS version 8.0 statistical software package .The average number of PCNA positive cells was counted across five fields of view among the thickest cartilage-cap area in each section by two independent observers under a magnification of × 400 . And 200 cells and PCNA positive cells were counted on the microscope counting line to determine the positive rate in each field. Finally, the average positive rate was used as the PCNA proliferation index. Statistical analysis was performed using non-parametric Wilcoxon rank sum test (Mann-Whitney U test) of two independent samples was used to compare the differences between the indicators of the two diseases, and EXT1 positive staining was located in cytoplasm, and the interpretation of EXT1 immunohistochemical results was based on Torlakovic EE’s method : the def


The effects of grinding (medium-coarse) and extraction time (14–22 h) on the physicochemical and sensorial properties of cold brew coffee produced using two types of Colombian specialty coffees (Huila and Nariño) were evaluated. Cold coffee brewed under coarse grinding and 22 h of extraction exhibited the highest values of total dissolved solids, extraction yield, pH, titratable acidity (TA), and total phenolic content. The type of coffee used mainly affected the TA and pH. All cold brew coffee samples had lower TA values than their hot counterparts. Nariño cold brew samples had higher TA values than those of Huila in all treatments evaluated. Higher scores were reported in the sensorial evaluation of cold brew coffee when prepared using the shortest time (14 h) and coarse grinding for both coffee types. These coffees were characterized by strong sweetness, fruity and floral flavours, medium bitterness and acidity, and a creamy body. Furans, pyrazines, ketones, aldehydes, pyrroles, esters, lactones, furanones, and phenols were detected as odour-active compounds. The findings of this study demonstrate that the particle size, contact time, and coffee type affect the physicochemical and sensorial characteristics of cold brew coffee, leading to cold brew coffees with different flavour profiles. The popularity of coffee arises from the stimulating effects of caffeine and from its pleasant aroma and flavour and, more recently, from its health benefits4. The characteristic coffee aroma and flavour are related to the volatile and non-volatile compounds generated during roasting that are brought to the cup by means of brewing5. Several metabolites present in the green beans during the harvesting and post-harvesting process contribute to the flavour after roasting and are associated with the quality of the coffee brew flavour. Since the 1990s, coffee quality has been related to the term “specialty coffee” or “café gourmet”, which refer to the highest quality of green coffee beans, with an excellent and unique flavor6. These beans have a known geographical origin and mostly include certified beans7.Every day, approximately 2.25 billion cups of coffee are consumed globally10. From an engineering viewpoint, coffee brewing is considered a solid-liquid extraction, where the roasted and ground coffee is in intimate contact with water. The water acts as a solvent to extract the water-soluble compounds, and depending on the extraction technique used, insoluble compounds may also be present in the extraction water as dissolved or suspended solids11. Finally, volatile and non-volatile flavour compounds are extracted from the ground coffee and are dispersed in the final coffee beverage12. In this process, the size reduction of the roasted beans by grinding is a prerequisite for the controlled extraction and dispersion of the chemical compounds in the coffee brew. Grinding is an operation that breaks down the roasted coffee into particles or smaller fragments. The formation of small particles, with a large particle surface area, is essential for the rapid liberation of carbon dioxide, the reduction of the diffusion distance for soluble substances during extraction, and for the improved transfer of colloidal substances to the liquid phase13.The majority of coffee beverages consumed are prepared by various hot brewing methods, depending on the geographic, cultural, and social context, as well as on personal preferences16.Due to the relevance of chemicals to the sensory characteristics of the coffee beverage, recent studies have focused on evaluating the effects of several variables along the entire production chain. In the literature, the most studied hot extraction methods are the espresso and filtered coffee methods. Studies have been carried to evaluate the influence of the product formulation  and brewing process variables  on the physicochemical attributes and sensory profiles17. Cold brew coffee can be made by dripping, direct or indirect immersion or by the French press method10.Numerous methods have emerged for coffee brewing, and most of them use high-temperature water (near the boiling point) and short times that do not exceed 5 minutes. However, in recent years, a new method has gained popularity in the market—the method of cold extraction. Cold brewing is carried out at room temperature (20 to 25 °C or colder) over a longer period than traditional hot brewing methods, with typical steeping times ranging from 8 to 24 hours18. Overall, a higher temperature increases the solubility19, and it affects the saturated vapour pressure of aroma compounds. Higher temperatures lead to a greater evaporation of organic volatile compounds (VOCs) and consequently to a higher release of these compounds. In cold extraction at room temperature, Fuller and Rao17 observed that the concentrations of 3-chlorogenic acid (3-CGA) and caffeine increased rapidly over the first 180 minutes before slowing and reaching equilibrium at approximately 400 minutes for different degrees of coffee roasting and grinding17.Low temperatures and long contact times produce a final coffee beverage with different physicochemical and sensory characteristics. These parameters affect the extraction speed and way of the chemical compounds present in the roasted coffee. The chemical compounds present in the coffee exhibit different chemical properties ; therefore, they have different extraction kinetics20. Several studies21 have compared different hot extraction methods and the physicochemical attributes and sensory profiles of these coffee brews. Conversely, despite the growing consumption of cold brew coffee and the interest in this coffee market, to the best of the authors’ knowledge few scientific studies regarding its physicochemical and sensory attributes have been published.Hot extraction has been widely studied, and it is well known that this brewing method affects the polyphenol extraction, caffeine content, total solids content, antioxidant activity, and the volatile compound profile22 found significant differences in the concentrations of non-volatile components such as caffeine, chlorogenic acid and trigonelline and in the flavour profile when they evaluated different extraction methods (dripping vs. stepping) and times . Higher concentrations of non-volatiles were found in the stepping method22. Non-volatile compounds have also been studied by Fuller and Rao17, who analysed the extraction kinetics and equilibrium concentrations of caffeine and 3-chlorogenic acid (3-CGA) in cold brew coffee compared with hot methods (French press) under different roast degree and grinding conditions. Higher concentrations of 3-CGA and caffeine were observed in cold brew coffee made with medium-roasted beans, and these compounds reached equilibrium at between 6 and 7 h of extraction, while 3-CGA concentrations and the pH were comparable between the cold and hot brews17. On the other hand, Lane and coworkers23 found that the caffeine concentration in cold brew coffee did not exhibit major differences compared to that of hot coffee23. Recently, Angeloni et al.24 measured the caffeine and cinnamic acid concentrations and some physicochemical and sensory characteristics in cold brew coffees that were prepared by using two cold brew methods. They reported that a higher temperature (22 °C) increased the total solids content, and the caffeine, caffeoylquinic acid, and 5-chlorogenic acid concentrations compared to the extractions carried out at 5 °C. Similarly, they found differences between the cold brew methods and temperatures evaluated regarding the flavour profiles, as measured in terms of bitterness, sweetness, sourness, and global intensity24.Regarding the cold extraction conditions and the final cold beverage composition, Kim and Kim25, some studies on cold brew coffee have evaluated bioactive compounds. For instance, Shin26 studied the chemical characteristics of polysaccharides isolated from cold brew coffee and found that cold brew coffee extracts could improve the functions of macrophages and the intestinal immune system. In the last year, Rao and Fuller27 evaluated the acidity and antioxidant activity of cold brew coffee made from light roast coffees of different coffee producing countries. They found that cold brew coffee extracts had lower concentrations of acidic compounds than hot brew coffee extracts prepared from the same beans and that the hot brew coffees had higher antioxidant capacities than their cold brew counterparts27.Due to more recent investigations that have indicated the potential health benefits of coffee beverage intakeCold brew coffee is a growing market within the coffee industry. In recent years, scientific interest has increased in this field. However, the effects of the cold extraction conditions on the chemical composition (non-volatile and volatile compounds) and sensory characteristics of cold brew coffee are still being studied. Therefore, this work analysed the extraction variables of extraction time, type of specialty coffee (Nariño and Huila), and grinding using cold brewing by the indirect immersion method. A sensory analysis was carried out to select the cold drinks most appreciated for their flavour compared with their hot counterparts. The selected cold brew coffees were analysed to determine their volatile organic compounds. This experimental configuration combining instrumental analysis with sensory evaluation to determine the volatile compounds and flavour characteristics of cold brew coffee has not yet been reported.> 0.05), Nariño cold brew samples showed higher values than Huila samples, mainly under coarse grinding  concentrations and total antioxidant activities compared with their hot brew counterparts27.The physicochemical characteristic results of cold brewed coffee under different extraction condition  are shown in Figs ing Fig. . Polyphe17 reported pH values between 5.40 and 5.63 for cold brew Coffea arabica (Kona Typica)17. In addition, Rao and Fuller27 found that the pH values of cold brew coffees of different coffee types (origin countries) ranged between 4.96 and 5.1327. Regarding total titratable acidity, the highest values (0.90 ± 0.04 mg/g chlorogenic acid) were found in the coarse ground and 22 h cold brew samples using Nariño coffee. Lower TA values (0.33 ± 0.03 mg/g chlorogenic acid) were identified in cold brew coffee with medium grinding and 14 h of extraction; these values were lower when Huila coffee was used.Although the grinding level and time had a significant effect (p < 0.05) on the pH and TA, these parameters were also influenced by the type of coffee used for cold brewing. The lowest pH values (more acidic) for Nariño and Huila coffees were 4.81 ± 0.01 and 4.88 ± 0.0, respectively, which were obtained under coarse grinding and 14 h of extraction for both coffees. Overall, higher pH values (less acidic) were reported for Huila coffee (4.92 ± 0.02), while the higher pH value from Nariño cold brew was 4.90 ± 0.0. Recently, Fuller and Rao27. In this work, physicochemical characteristics of cold and hot brews were evaluated when prepared using a similar ratio of coffee/water. The results are shown in Table Commercial vendors and coffee enthusiasts often suggest that cold brew and hot brew coffees have different taste profiles, acidity levels, and caffeine contentset al.29 found a higher content of total solids and caffeine with a prolonged extraction time under hot extraction. A longer brewing time allows increased extraction of some compounds9, but these compounds can be highly susceptible to oxidation and degradation30.Zanoni 31. Besides, the concentration of these compounds in roasted coffee beans vary owing to the coffee’s origin, post-harvest processing, roasting, and storage32. These facts could explain the difference in the phenolic concentration observed between types of coffees and extractions ways.Hot coffee brews showed significant differences between them regarding TA and TPC. Higher values for these variables were found in the brews made with Nariño coffee . In cold brew coffee, the highest TPC values were found in treatments that used Nariño coffee, coarse grinding and 22 h of extraction . This value was comparable with the TPC found in the hot brews  made with the same coffee. The differences in the TPC values can be explained because there are several water-soluble polyphenols present in roasted coffee which exhibit different chemical properties, some of them such as chlorogenic acids, caffeine, and soluble melanoidins are removed under different contact times27 reported that the titratable acidity in hot coffee extracts was larger than that measured in cold brew coffee extracts, which could indicate that hot brewing was able to extract more acids and additional acidic compounds27. Moreover, several studies have revealed that the pH and TA are dependent on many factors, such as the geographic origin of the green coffee beans, fruit maturity, harvesting processes, weather conditions during harvesting and drying, and all steps in the post-harvest processing. Furthermore, pH and acidity have been associated with the type of roasting process and brewing method34.In this study, the pH values of the cold brew coffees were significantly different (p < 0.05) from the hot brews. Cold brew coffees exhibited higher pH values (less acidic) than their hot counterparts. In the hot beverages, the total acidity was higher than in all of the cold brew coffees. Total acidity in the hot beverages was higher than in the cold brew coffee. Recently, Rao and Fuller35. The results from the sensory analysis for the cold and hot brew coffees are shown in Tables In this study, the flavour profile of the coffee brew was evaluated regarding its general characteristics and the intensity of specific attributes that are commonly used in the coffee industry. Global characteristics include terms such as fragrance and aroma, acidity, body, flavour, and aftertaste, while the attributes are specific descriptors that may not occur in every cup of coffee, but when they are present can add complexity and uniqueness to the coffee36. Consequently, the assessors showed that although some flavour characteristics in cold brew coffees were less intense compared to the hot beverage, the taste of the cold drinks, according to their impression , was acceptable for a coffee beverage. The best scores for cold brewed coffees in terms of overall impact were for those samples brewed using coarse grinding and 14 h of extraction for both coffee types.Regarding global flavour characteristics, the data in Table The results in Table 10. In the scientific literature, there are limited data regarding the flavour profile of coffee brewed under different extraction conditions. Kim and Kim22 found differences in flavour in terms of chocolate, winey, acidity, sweetness, bitterness, body, and aftertaste attributes when stepping and dripping methods were used. They found that cold brew coffee at 18 h was the most acceptable to consumers according to the flavour profile22. Recently, Angeloni and coworkers24 reported that the flavour can vary depending on the cold brew method (dripping and immersion), but in general they found that cold brew coffees can exhibit higher intensities of sugar caramelization, sweetness, and bitterness24.Overall, cold brew coffee is typically considered to be sweet, with chocolate and syrupy notes37. Thus, in the present study, the cold brew coffees samples with the best scores in the global sensorial characteristics were selected to determine their volatile compound profiles. The cold beverages prepared with Nariño and Huila coffees under coarse grinding conditions and 14 h of extraction were selected. The volatile compounds in the cold extracts were identified and quantified through GC–MS  furans (150), pyrazines (100), phenols and ketones (90 in each class), pyrroles (80), hydrocarbons (76), carboxylic acids (60), esters (55), alcohols (50), and aldehydes (45). Clearly, not all of the approximately 1000 volatile compounds are relevant in terms of aroma. The literature suggests that only approximately 5% of these compounds may be responsible for the aroma of coffee, which would result in approximately 50 odorant compounds38 in roasted coffee. However, the presence of volatile compounds in the final coffee brew depends on the coffee bean chemical compounds, the roasting process, and the extraction technique. According to López-Galilea, et al.39, four classes of compounds appear to have a high impact in the aroma of coffee: pyrazines, furans, aldehydes, and ketones39. The current results showed that most of the volatile compounds detected in both of the cold coffee samples belonged to these chemical classes.The volatile compound profiles were qualitatively and semi-quantitatively analysed (relation of area percentage from GC data). The highest numbers of volatile compounds (in order of abundance) were furans, pyrazines, ketones, aldehydes, pyrroles, esters, and acids for both cold brew coffee samples. Phenols were only found in the Nariño cold brew coffee, while two acids and pyridine were only detected in the Huila cold brew coffee. Regarding volatile compounds, Flament40. In general, furans were the major class of volatile compounds detected in the cold brew coffee samples. Furans exhibit malty and sweet roasted aromas with relatively high sensory thresholds compared to other volatile groups found in coffee32. These characteristics could be related to the sensory attributes described by the assessors who found that the cold brew coffees had malt and sweet flavour attributes. Although similar compounds in the furans group were found in the two cold brew coffee beverages, the relative areas of certain compounds in the Nariño sample were higher, mainly for 5-methylfurfural (11.84% Nariño and 7.03% Huila), which has been associated with almond notes, caramel, and burnt sugar41.Figure 12. In general, pyrazines have been described as having nutty, earthy, roasty, and green aromas. They are most noticeable at higher serving temperatures39. Despite the low temperatures used during cold brewing, the samples analysed in this study showed flavour attributes associated with pyrazines when they were assessed by the judges. Ketones were also one of the three most abundant groups in both of the cold brew coffee samples. According to López-Galilea, et al.39, ketones are one of the most abundant compounds in filtered coffee brews. Seven ketones were identified in the Huila cold brew coffee, while only four were found in the Nariño cold brew. Ketones have been described as having buttery, caramel-like, musty, mushroom-like or fruity odour notes39. The cold brew coffee also presented aldehydes in their volatile profiles. 2-Methyl butanal and 3-methyl butanal showed high concentrations for both coffees (Huila and Nariño) -furanone was only presented in the Nariño cold brew coffee  that are applied in cold extraction tended to increase the values of TDS, EY and TPC, which were even higher when coarse grinding was used. In the extraction process, temperature can be considered as the driving force that favours the extraction of the chemical compounds present in the coffee grounds. At higher temperatures, the kinetic energy of the water molecules is higher, leading to a faster removal of the numerous compounds present in the roasted coffee. Conversely, during cold coffee brewing, the temperature is low (less than room temperature); therefore, the extraction and diffusion of several compounds requires more time to compensate for the low temperature. This could explain the fact that the cold brewed coffee with longer extraction times exhibited EY, TDS, and TPC values close to or even higher than their hot counterparts.27. All cold brew samples evaluated in this study showed lower TA values than their hot counterparts. Recently, similar results have been reported by Rao & Fuller27 in cold brewing with coffees from different countries. These differences suggest that the cold and hot brew coffees are similar in their total concentrations of deprotonated acid compounds, but differ in the concentration, and possibly in the complexity, of protonated acids at the pH of extraction27. Several acids such as acetic, formic, malic, lactic and phosphoric, along with quinic and chlorogenic acids, have been detected in coffee brews52. Some of these can be extracted very efficiently in the initial seconds because their solubility increases with temperature, which could be why the hot brews exhibited higher TA values than the cold brews.In addition to the effect of grinding and extraction time, the type of coffee also had a significant effect on the pH and titratable acidity (TA). Measurements of pH quantify the concentration of aqueous hydrogen ions at the time of analysis, providing a metric for the quantity of deprotonated acid molecules in a sample. Total titratable acidity is a measure of all acidic protons in a sample, including non-dissociated protons52 found that in hot coffee brewing, over-extraction incorporated undesirable and less soluble aromatic compounds into the drink.Although the coarse grinding and longest extraction time were identified as the optimal conditions for the physicochemical properties, higher scores in the global sensorial evaluation were reported for samples processed using coarse grinding and the lowest extraction time (14 h). This may be because lengthy extraction times can lead to over-extraction of the compounds associated with undesirable sensory notes. Likewise, lengthy extraction times may lead to the degradation of compounds in the beverage, and off-flavour compounds may be formed in the cold brew coffees. Illy and Viani27. However, there are few published scientific studies that have carried out a cold brew flavour analysis. The results of this work revealed that cold brew coffees present global flavour profiles with less intensity in the aroma, taste, aftertaste, acidity, and body than their hot counterparts. The global perception or overall impact indicated that the assessors found that cold brew coffees had an acceptable flavour. The complexity of the cold brew coffee flavour also showed that there are specific attributes that are different from a hot brew. Cold brews presented the highest or similar values when compared to their hot counterparts for the flavour attribute scores of malt, pepper, almond, cocoa, chocolate, floral, bitterness, sweetness, and smooth and creamy body. Many of these had higher scores when a 14 h cold extraction was employed.Several coffee vendors have marketed cold brew coffee with claims related to tasting smoother and less bitter and having a sweet taste and low acidity38.According to volatile profile results, both coffees presented differences in the number of compounds identified. As yet, there are no studies regarding the volatile compounds of cold brew coffees that allow making a rigorous comparison. In general, the high relative concentration of furans, pyrazines, and ketones in both samples (Nariño and Huila) could be associated with sweetness, malty and fruity, which was also described by the assessors in the sensorial analysis. Previous studies have indicated that the aroma of the filtered beverage is associated with the aromas of the alkylpyrazines, phenols, furanones and some thiols. These volatile compounds have been generally associated with sweet, caramel, herbal, fruity, malty, and nutty flavorsThis research reports that some physicochemical characteristics, such as extraction yield, total dissolved solids, total phenolic content, pH and titratable acidity, can be strongly affected by the grinding level of coffee beans. When studying the role of grinding in the chemical composition of cold brew coffee, it is essential to consider the particle size distribution and coffee packing bed and their complex relationship with the diffusion and mass transfer phenomena. In cold extraction, a long contact time (22 h) increased the extraction yield, total dissolved solids, total phenolic content and titratable acidity. The type of coffee mainly affected the titratable acidity and pH. The Nariño cold brew samples showed titratable acidity values higher than those of Huila. Overall, all cold brew coffee treatments showed lower values of titratable acidity than their hot brew counterparts. The general characteristics of the cold brew flavour were less intense than those of their hot brew counterparts. According to the maximum overall sensory impression during the entire tasting time, the cold brewed coffee flavour was typical for this type of beverage.The cold brew volatile profile identified in both coffees was related to some flavour attributes found in the sensorial analysis . These results open new insights for future investigations related to the identification and quantification of volatile and non-volatile compounds and their relationship to the cold brew flavour profile under different extraction conditions.Coffea arabica var. caturra) from two origins, Huila and Nariño (Colombia), were used in all trials. The coffee beans corresponded to a micro-lot, which was harvest and processed by smallholder coffee growers in 2017. The roasted coffee beans from the Huila and Nariño regions of a medium-roasted degree were sourced from the local coffee store Cafecultor® . The roasted coffee beans were ground in a commercial mill  at two levels: coarse and medium. The particle size distributions of each grinding level were determined according to NTC 2441, Colombian Technical Standard, where the average of the particle size was determined by granulometric distribution53. An average particle size of 501 to 700 μm was used in medium grinding and 701–900 μm in the coarse grinding.Arabica coffee , extraction time (14 or 22 h), and type of specialty coffee (Huila or Nariño) were studied. The experiments were carried out following a randomized design with a factorial structure (2 × 2 × 2) in duplicate. The measurements were carried out in triplicate. The extracts were stored in amber glass bottles at 4 °C until analysis. Hot brew extraction was performed according to Gloess, et al.9, with certain modifications; 42.5 g of coffee in 500 g of water was used to reach a coffee/water ratio similar to that in the cold brewing. French press coffee was prepared by pouring boiling water onto coffee grounds in a glass French press (Bodum®).The cold brewing process was carried out at room temperature (20 °C) using the immersion method with filtered water, according to Fuller and Raoet al.54, where defines the relationship between the concentration of soluble solids (Xs) and the Brix degrees as follow: XS = 0.0087 °Brix54. The EY was calculated as the relationship between the TDS, total weight of extract obtained (Wb), and ground coffee weight used in the extraction (Wgc), defined by the following equation5: EY (%) = (TDS * Wb/Wgc) * 100.The TDS were measured following the methodology proposed by Moreno, 55.The pH value of the cold brew samples was measured at 19 °C using a pH-meter . For the total TA, 50 mL of the coffee extract was titrated with 0.1 mol/L NaOH solution at a pH of 6.5, and the result was expressed in milligrams of chlorogenic acids per gram of coffee (NTC 5247)56, with certain modifications57. Each sample was diluted 1:100 with distilled water. Thereafter, 90 µL distilled water, 15 µL sample, 7,5 µL Folin–Ciocalteu reagent , and 22.5 µL of 7.5% w/v sodium carbonate were added to each microplate, followed by the addition of 15 µL distilled water. All samples were incubated for 2 h at room temperature before measuring the absorbance at 750 nm. Each sample was measured in triplicate. The results were expressed in grams of gallic acid equivalents per liter of solution.TPC measurements were conducted by means of the Folin–Ciocalteu method described by Singleton and Rossi36 for coffee quality. Likewise, the panel selection was conducted according to requirements listed in ISO 8586-158. Prior to the assessment of the cold brew coffee samples, the assessors participated in additional sessions to develop a sensory vocabulary for the cold coffee beverages. These samples were composed according to the Colombian Technical Standard NTC 393259 and ISO 1103560. The initial list of attributes was classified into six categories: aroma, taste, aftertaste, acidity, body/mouthfeel, and overall impact. In each category, sensory descriptors were included according to those described by Chambers et al.35, with certain modifications. The tasters were trained for more than 40 h in cold brew coffee tasting, with the purpose of increasing their experience in the use of the vocabulary.Sensorial analysis of the coffee samples was carried out by a trained panel of three tasters , with more than three years of experience in the area of coffee sensory analysis. All judges had a broad experience and training in the cupping process established the SCAA protocol59. Each cold brew coffee sample was served in three replicates and was tested at room temperature (19 °C). The hot brews were assessed at 70 °C61, also in triplicate. The experimental design was made in two blocks, and the sessions for sensory analysis were performed on different days. The samples were analysed immediately after the completion of the extraction time. The cold and hot brews were evaluated in different sessions, and the sample order was randomized within each session and block.During the first stage, the sensory features of the cold and hot coffee samples were evaluated on a zero to five scale, using only the general groups of sensory descriptors . Within the scale, 3 corresponded to an attribute that was considered to be good, 3.5 was very good, 4 was excellent, and above 4.5 was outstanding. The panellists then evaluated the intensity of the specific attributes in each category. According to the range, five (5) was equivalent to a very strong attribute intensity, while zero (0) was not perceptible et al.62, with certain modifications. Each sample (2 g) was equilibrated for 1 h in a 40 mL sealed vial at 18 °C using a magnetic stirrer. The volatile compounds released from the headspace of each sample were collected using 65 µm PDMS/DVB 1 cm long fiber  for 1 h at 18 °C and then directly injected (5 min of fiber desorption time at 250 °C) into a gas chromatograph, Agilent 7890B, coupled to a 5977A mass selective detector . A DB-FFAP column  was used. The column oven was programmed from 45 (after 2 min) to 250 °C at 5 °C/min, and the final temperature was maintained for 5 min. The injector temperature was maintained at 250 °C; the carrier gas was 1.5 mL of He/min. Structural elucidation of the volatile compounds was performed by comparison of their mass spectra and retention indexes , with those of either the standards or NIST mass spectra library . Data were processed by the Class 5000 v 2.2 MS-Workstation software.The volatile compounds of the cold brew coffee were obtained by Headspace–Solid-Phase Microextraction (HS-SPME) and analyzed by gas chromatography coupled with mass spectrometry (GC–MS), according to the method reported by Moreno, All experiments were carried out following relevant guidelines and regulations of Universidad de La Sabana. The Research Commission of the Faculty of Engineering of the Universidad de La Sabana approved the procedures and methodologies related to the sensory analysis of the coffee extracts. All participants of the sensory analysis were volunteers and signed an informed consent form with the type of the research and agreement to develop the sensory analysis of the coffee extracts produced under the conditions described in the section “sensory evaluation of cold and hot brew coffee.”The effects of the particle size, extraction time, and coffee origin on the physicochemical variables and sensory attributes were evaluated using a randomized block factorial design. One-way analysis of variance was applied to determine the difference between treatments, followed by an LSD test with a significance level of 95% using XLStat version 2018.1 .