High metabolic load distance provides global information about the soccer players’ total high-intensity activities. Thus, this study aimed to examine the Spanish professional soccer players’ high metabolic load distance profile, comparing competitive level and playing positions.−1 and accelerations or decelerations  were included), and HMLD per minute (HMLDmin) were analyzed by the ChryonHego® video-tracking system. Players were classified according to their playing position as follows: Central Backs (CB), Full Backs (FB), Center Midfields (CM), Wide Midfields (WM), and Forwards (FW).A total of 18,131 individual match observations were collected from outfield players competing during the 2018/2019 and 2019/20 seasons in the First and Second Spanish Professional Soccer Leagues (LaLiga™). High Metabolic Load Distance (HMLD; distance covered with a power consumption above 25.5 W·kgmin overall and during the second half. However, when playing positions were considered, differences between competitive levels were observed in all positions, mainly in HMLD and HMLD during the first-half variables. In addition, several differences between playing positions were observed, with CB presenting the lowest values in all variables compared to their counterparts in both competitive levels, whereas CM in First Division and WM in Second Division showed the highest values in the HMLD variables.No differences between competitive levels were found in any variable when all players were analyzed conjointly except for HMLDThe findings are of interest to analyze the HMLD in professional soccer players, enabling the adaptation and individualization of training in this population according to the competitive level and specific playing position of each player. In this−1  also are included in HMLD. This variable provides global information about the players’ total high-intensity activities, as it not only includes high velocity running but also accelerations and decelerations, justifying its analysis in soccer . In addition, these authors also reported that forwards achieved the highest HMLD values  compared to their counterparts. Recent studies have been conducted focusing on external demands in professional soccer players including HMLD variables. Specifically, To address the gap identified in the literature, we analyzed the external demands, in terms of HMLD, of Spanish male professional soccer players during two competitive seasons (from 2018/19 to 2019/20). Specifically, this study aimed to examine the Spanish professional soccer players’ HMLD profile, comparing competitive level and playing positions. Based on previous studies , we hypoi.e., a match day too much rainy, windy, or snowy) during the match that hindered accurate data collection, so 18,131 match observations were analyzed . Players were classified according to their playing position as follows: Central Backs (CB), Full Backs (FB), Center Midfields (CM), Wide Midfields (WM), and Forwards (FW)  during the 2018/2019 and 2019/20 seasons. Goalkeepers and players who did not play the full game were excluded from the analysis. Thus, 17,077 recordings were excluded due to inclusion criteria, issues related to repeated signal loss by the system, or adverse weather conditions (rds (FW) . Data wei.e., 2018/2019 and 2019/2020), resulting in 18,131 match observations being collected. All the study variables were collected through the optical tracking system ChyronHego® , which is composed of eight super 4K-High Dynamic Range cameras based on a positioning system (Tracab – ChyronHego VTS). This system records from several angles and analyzes X and Y positions for each player, providing real-time two-dimensional tracking (acquisition frequency = 25 Hz). The validity and reliability of the Tracab® video tracking system have been analyzed, reporting average measurement errors of 2% for the physical performance  as the main one. This variable refers to the distance covered with a power consumption above 25.5 W·kg−1. This value corresponds to running at a constant velocity of 5.5 m·s−1 or 19.8 km·h−1 on grass. Accelerations or decelerations  also are included in HMLD. This variable provides global information on the players’ total high-intensity activities, as it not only includes high velocity running, but also accelerations and decelerations. Specifically, relative values of HMLD were calculated: HMLD during the first half of the matches (HMLD1); HMLD during the second half of the matches (HMLD2); HMLD per minute (HMLDmin); HMLD during the first half of the matches per minute (HMLD1min); and HMLD during the second half of the matches per minute (HMLD2min).All the variables included were recorded by Mediacoachi.e., each player has a record for any match played). To determine the adequacy of this statistical procedure, we first calculated the levels of within-person variance for each player by constructing unconditional null models. These unconditional models allowed us to calculate the Intraclass Correlation Coefficient (ICC), which presented values greater than 10%, indicating the existence of variability in the data and justifying this analysis approach . No significant differences between competitive levels were found for any variable except for HMLDmin and HMLDmin2. Conditional r2 values ranged from 0.54 to 0.64 in all models showing a moderate effect of competitive level on match physical demands.p < 0.001). Significant differences were also observed when First Division CB were compared with Second Division CB in the HMLD, HMLD1, and HMLD1min variables (p < 0.001). Regarding FB, significant differences with CM were observed in all variables (p < 0.05) except for the HMLD2 in the Second Division. Differences between FB and WM were also observed in all variables (p < 0.05) except for HMLD and HMLD2 in the First Division. Differences between FB and FW (p < 0.05) were found in HMLD, HMLD1 (only in the First Division), HMLD2, HMLD1min, and HMLD2min (only in the Second Division). Moreover, significant differences were observed when FW in the First Division were compared with the FW in the Second Division in HMLD and HMLD1 variables (p < 0.05). CM presented significant differences (p < 0.05) with WM in all variables except for HMLDmin and HMLD2min, whereas differences in HMLD1min were observed only in the First Division. Significant differences between CM and FW were observed in all variables and levels (p < 0.05). In all variables, CM in the First Division presented higher values compared to CM in the Second Division (p < 0.001). Attending to WM, significant differences with FW were found in all variables and levels (p < 0.01). Significant differences between WM in both levels were also found in HMLD and HMLD2 (p < 0.05). Significant differences between FW in both levels were only observed in HMLD1min (p < 0.05). Finally, conditional r2 values ranged from 0.54 to 0.64 in all models showing a moderate effect of playing position on match physical demands.Differences according to playing positions are shown in min and HMLDmin2. However, when playing positions were considered, differences between competitive levels were observed in all positions, mainly in the HMLD and HMLD1 variables. Several differences between playing positions were also observed, with CB presenting the lowest values in all variables compared to their counterparts in both competitive levels, whereas CM in the First Division and WM in the Second Division showed the highest values in the HMLD variables.The main aim of this study was to examine Spanish professional soccer players’ HMLD profile, comparing competitive level and playing positions. This is the first research that compares the match physical demands of competitive level and playing positions attending to this variable, which reflects the high-intensity profile of soccer players. No differences between competitive levels were found in any variable when all players were analyzed conjointly except for HMLDmin and HMLDmin2. On the contrary, previous research have showed differences in match physical demands between soccer leagues. For example, the Premier League soccer players covered less total distance and high intensity running distances than the lower leagues players  in all the passages analyzed  compared to their counterparts. e.g., situations near to the goal without longer distances covered at high intensity), which favors their HMLD values being significantly lower than those of the rest of the playing positions. On the other hand, the highest HMLD distances are covered by different playing positions as a function of competitive level, with CM players obtaining the highest values in the First Division, whereas the highest values in the Second Division were observed in WM players. These differences between competitive levels could be related to the differences in the playing styles followed in each division (Previous studies have determined that professional soccer players’ external demands during match-play vary according to their specific playing position . Therefodivision . Howeverdivision , WM presdivision . FinallyThis study is not without limitations. Firstly, this investigation was conducted only in the top two Spanish professional soccer leagues, so the findings obtained cannot be extrapolated to different leagues, as external demands vary according to the country. Secondly, the teams were not differentiated according to different contextual variables, such as the match location, the quality of the opponent, or the playing style. In addition, would be interesting considered the match status  and the min and HMLDmin2. Differences between competitive levels were obtained in all positions when analyzed isolatedly, mainly in the HMLD and HMLD1 variables. Several differences between playing positions were also observed, with CB presenting the lowest values in all variables compared to their counterparts in both levels, whereas CM in the First Division and WM in the Second Division obtained the highest values in the HMLD variables. In practical terms, this study could serve as a reference for the analysis of HMLD in professional soccer players, improving the adaptation and individualization of the training process in this profile of team sport athletes according to the competitive level and specific playing position of each player.In conclusion, this study has confirmed the lack of HMLD differences between competitive levels when all the players were considered conjointly, except for HMLD10.7717/peerj.13318/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13318/supp-2Supplemental Information 2Click here for additional data file.


TM swabs were used to collect 100 cm2 area samples at seven different locations throughout the beef processing line at four different regions on the carcass. Each of the eight sampling days evaluated included three samples collected per sampling location/carcass region for a total of 84 samples per day. Enumeration of total aerobic bacteria, Enterobacteriaceae, and Escherichia coli was performed on each sample. Microbial SPC parameters were estimated for each sampling point. Statistical differences between sampling points for all carcass locations (p < 0.001) followed an overall trend with higher values at pre- and post-evisceration with a continuous decrease until final interventions with a slight increase in counts during the chilling process and a final increase after fabrication. Variability at sampling points is the result of the nature of the process and highlights open opportunities for improvement of the food safety system. Microbial baselines and SPC parameters will help support decision making for continuous process improvement, validation of intervention schemes, and corrective action implementation for food safety management.The objective was to conduct a bio-mapping of microbial indicators to determine statistical process control (SPC) parameters at a beef processing plant to establish microbiological baselines and process control parameters to support food safety management decisions. EZ-Reach Escherichia coli O157:H7 outbreak in United States ground beef during 1992–1993, the meat industry and regulatory agencies changed completely the way of processing and inspection of red meat. The U.S. industry and government focused their attention on reducing the risk of pathogen presence on carcasses and products that can cause foodborne illness due to the consumption of red meats. According to the Interagency Food Safety Analytics Collaboration (IFSAC), in 2012 the attribution of foodborne illnesses to Escherichia coli O157:H7 from beef was close to 46%, being the highest contributor for that year [Ever since the Escherichia coli O157:H7 [Escherichia coli O157:H7 in ground beef and trim, the mandated implementation of the Hazard Analysis and Critical Control Point (HACCP) systems, the addition of six non-O157 Shiga-Toxin producing Escherichia coli serogroups to the list of adulterants in beef, and the discussion about potential new performance standards for Salmonella spp. are some of the key events that incentivize the U.S beef industry to continuously evaluate and implement the use of antimicrobial interventions during the beef harvest [Multiple aspects have changed in the regulatory requirements established by the U.S. Department of Agriculture’s Food Safety and Inspection Service (USDA-FSIS) to control the presence of  O157:H7 . The zer harvest . Antimic harvest ,10,11,12Live cattle are carriers of pathogenic microorganisms which later can contaminate carcasses during slaughter in commercial processing settings. Moreover, beef is an animal product that features adequate conditions like pH, water activity and nutrient content to not only become contaminated but also to support the growth of these microorganisms during storage and handling that may result in important public health threats. Researchers have indicated that there are three key issues in the production of meat products that have an importance on the risk of contamination: (1) pathogen levels on hides, (2) correct removal of hides minimizing cross-contamination between hides and carcass, and 3) efficacy of antimicrobial interventions during the slaughtering process [ efficacyMultiple factors can affect indicator and pathogen levels on hides such as the season, where higher levels of pathogens can be found during warmer months, or the production system, where animals on feedlots, due to closeness between the animals, present higher fecal contamination on hides when compared with animals on grazing systems . DespiteThe efficacy of antimicrobial interventions during the slaughtering process is one of the key drivers for pathogen reduction during production of meat in the United States. Consumers expect that foods in general are free of foodborne pathogens and have a reasonable shelf-life, where interventions play a huge role . AccordiBio-mappings of microbial indicators and determinations of statistical process control parameters are proven tools that beef processing operations have available to evaluate the performance of their sanitary practices and interventions, and to investigate if their process is under control ,21. By cThe goal of this study was to compare microbial data collected periodically with historical data and determine if the process variation is in control (consistent) or if it is out of control (unpredictable) . BecauseSwabs collected for this study were obtained from a large commercial USDA-FSIS inspected beef processing facility located in the U.S. This plant operates in two shifts and processes around 1800 heads of cattle per day. During beef slaughter, the microbial interventions applied consist of a steam vacuum after de-hiding at the inside round area, hock, and shank with steam at 82 ± 5 °C, application of a carcass water wash at room temperature before evisceration (pre-evisceration), followed by a 2–5% concentration lactic acid spray treatment at 49 ± 5 °C applied in a uniform spray pattern for coverage of the whole carcass at a pressure >15 psi. After evisceration and splitting (post-evisceration), a carcass water wash at room temperature was applied, followed by a lactic acid spray treatment at 2–5% concentration at 49 ± 5 °C, applied in a cabinet at a pressure >15 psi. Subsequently, trimming and visual inspection along the line is performed  until reaching a hot water whole carcass wash at 85 ± 5 °C plus a lactic acid spray treatment (2–5%) at 49 ± 5 °C applied in a uniform spray pattern in the whole carcass at a pressure >15 psi (after interventions). After final interventions, carcasses are held in a cooler for 18–24 h, reaching a surface temperature at the end of the chilling period of <7 °C (after chilling). Then, carcasses go through a lactic acid spray cabinet (2–5%) with an operating pressure >15 psi and temperature around 49 ± 5 °C while exiting the holding cooler and prior to the entry into the fabrication room (cold carcass). Finally, a lactic acid spray (2–5%) with an operating pressure >15 psi and temperature around 49 ± 5 °C is applied on each of the different sub-primals processing lines (after fabrication). A total of 7 locations in the plant were evaluated.TM cattle 100 cm2 template  at each of the seven different sampling locations in the beef processing line  on four carcass regions . Each sampling day included three samples per sampling location/carcass region, for 7 sampling locations and 4 carcass regions, resulting in a total of 84 samples per day. Samples were collected during eight different days of processing, equally spaced in two months, to account for lot variations, intrinsic process variability, and statistical process control analysis during summer 2021. Sampling locations were determined due to the highest likelihood of contamination or the presence of an intervention step in the process.EZ-Reach™ 25 mL buffered peptone water (BPW) swabs  were collected using a 3MEscherichia coli (EC). Samples were enumerated using the TEMPO® system  following the AOAC 121.204 official method for enumeration of aerobic plate counts, AOAC 050.801 for Enterobacteriaceae, and AOAC 080.603 for Escherichia coli. All cards were incubated at 35 ± 1 °C for 24 ± 2 h.Swabs were immediately chilled and shipped overnight to the ICFIE Food Microbiology Laboratory at Texas Tech University for microbiological analysis. Upon arrival, swabs were homogenized in a stomacher  at 230 rpm for one minute. Homogenates were serially diluted in 9 mL BPW  tubes and plated for aerobic plate counts (APC), Enterobacteriaceae (EB), and 2 for APC and Log CFU/sample for EB and EC to facilitate data visualization. A one-way ANOVA analysis was performed, comparing counts at each of the different steps in the beef processing line for each carcass region, followed by pairwise comparison T-test adjusted by the Benjamin and Hochber method. If parametric assumptions were not met, the Kruskal–Wallis test was used as a non-parametric alternative for the ANOVA, followed by a pairwise multiple comparison Wilcoxon’s test adjusted by the Benjamin and Hochber method. All significant differences were evaluated using a p-value lower than 0.05.All data were analyzed using R (Version 4.1.2) statistical software to evaluate the change in microbial loads at different process locations  throughout the beef processing line for each of the four carcass regions . Counts were transformed into Log CFU/cm3 factor (0.866), given that 12 samples were collected on each sampling day during 8 eight days [Furthermore, the Shewart’s expected average statistical quality control chart ,  at the end of the slaughtering process (Cold Carcass) when compared with pre-evisceration, with reductions of 1.16, 1.68, 1.64, and 1.15 Log CFU/cm2 for shank, inside round, midline, and brisket, respectively, , . An overectively . Aerobic CFU/cm2 . Counts ctively, , , ((p = 0.002), being the highest at shank and followed by inside round and brisket. The lowest counts found during the entire process were after final interventions (After Interventions) with 1.65, 1.47, 1.53, and 1.79 Log CFU/sample for shank, inside round, midline, and brisket, ((p < 0.001) at the end of the slaughtering process (Cold Carcass) when compared with pre-evisceration, with reductions of 0.51 and 0.78 Log CFU/sample for inside round and midline, while no difference was found for shank and brisket . When assumptions are not met for performing parametrical analysis, a Kruskal–Wallis test was preferred instead of ANOVA test to find differences between sampling points throughout the beef processing line. Interventions and dressing practices throughout the beef processing line significantly changed the Enterobacteriaceae counts at each of the four different carcass regions  0.001), . A simil 0.001), . Enterobbrisket, . Counts  brisket , , (Escherichia coli counts at the midline region of the carcass at pre-evisceration were significantly lower than shank and inside round regions (p = 0.001), being the highest at shank and followed by inside round, and brisket. The lowest counts found during the entire process were after final interventions (After Interventions) with 1.40, 1.45, 1.46, and 1.41 Log CFU/sample for shank, inside round, midline. and brisket, ((p < 0.001) at the end of the slaughtering process (Cold Carcass) when compared with pre-evisceration, with reductions of 1.18, 0.49, 0.34, 0.74 Log CFU/sample for shank, inside round midline, and brisket (For  0.001), . A simil 0.001), . Escheribrisket, . Counts  brisket ,  but in later steps were able to growth due to recovery when collecting the post-chill sample. Nonetheless, this increase it can also be attributed to re-contamination at the hot box or during fabrication due to the manipulation of the carcass or simply by the ineffectiveness of subsequent interventions.Statistical process control charts have a very close relation with hypothesis testing. The null hypothesis is that a process is under control while the alternative hypothesis is that the process is not under control. A point outside the limits established by the UCL and LCL can be considered as evidence against the null hypothesis but at the same time, it is also possible that this point is just error generated by random chance. The average run length (ARL) can describe the frequency with which theses errors occur. In a process that is under control, the probability that a point is outside the control limits (±3σ) is equal to 0.0027 (0.00135 + 0.00135); therefore, the ARL will be 370.4 (1/0.0027), meaning that it can be expected to find one sample every 370 samples that will plot outside the control limits just by random chance. In our examples, few observations are outside the UCL–LCL range which may lead to the conclusion that some of the steps are under control, but only 12 observations were collected during eight days. Therefore, more samples collected over longer time periods should be included in the analysis to make inferences that the process is under control, but it will be expected that if the process is out of control, the ARL will be less than 370.4.The comprehensive enumeration of microbial indicators throughout different steps in beef processing facilities (bio-mapping) is a novel approach for processors to have a general overview of how well their process performs in terms of achieving and maintaining low microbial loads. Bio-mapping studies allow processors to visualize upon data analysis the overall picture of which steps in the process are the most important for ongoing monitoring due to their effectiveness, and which ones can be removed or modified as their effect does not contribute to the overall reduction of microbial counts. Besides, microbial baselines developed by bio-mapping allows processors to make decisions based on ongoing comparisons with historic data of the process when process modifications are considered for implementation, including new processing schemes, new antimicrobials, or adjustments in speed lines, among others.Escherichia coli yielded reductions during the slaughter process in ranges of 1.7 to 2.44 Log CFU/cm2, 1.04 to 1.82 Log CFU/sample, and 0.41 to 1.43 Log CFU/sample, respectively. The results support the importance of using multiple-sequential interventions in beef packaging plants as a way to improve the microbiological quality of beef carcasses [Escherichia coli O157:H7 and Salmonella typhimurium rather than when used individually [2 on beef trim in a commercial plant environment [Escherichia coli by 0.6 Log CFU/cm2 more than using the steam pasteurization or the water wash by itself [In this study, a clear trend for all indicator microorganisms for most of the different carcass regions sampled was observed with microbial reductions from pre-evisceration until final interventions, followed by an increment during chilling and fabrication. Enumeration of aerobic plate counts, Enterobacteriaceae, and arcasses ,24,25,26arcasses . The usevidually . The comironment . Furthery itself . Since ty itself ,30,31.Escherichia coli counts were found in the brisket region of cold carcass, and for all indicator microorganisms, the highest counts were detected in the inside round after fabrication. This information provides a foundation to make decisions in support of food safety management programs, such as which carcass regions should be considered for sampling to analyze worst case scenarios, re-evaluation of interventions in the process, identification of key steps in which minimum changes may lead to dramatic non-compliance microbial standards in final product, or simply the understanding of increases in microbial levels due to the nature of the process in specific regions of the carcass. Furthermore, if there is an established sampling program and adequate analysis of the microbial data, this information can result in the implementation of targeted strategies such as antimicrobial interventions in specific areas of the carcass or different antimicrobial concentrations according to the region fabricated, that are based on risk and the likelihood of presence of high or low levels of microbial counts [Bio-mapping can also facilitate processors in understanding the performance of the process and can help the food safety team make data-based decisions on quality and safety aspects of beef meat. For all indicator microorganisms, swabs collected in the shank at pre-evisceration resulted in the higher counts; conversely, after evisceration, samples of the midline region presented the highest counts. The lowest counts during the process were observed after final interventions during slaughtering. The highest l counts .In addition to bio-mapping studies, processors have the opportunity to use the microbial counts data to statistical process control parameters, as a tool for microbial quality assurance in beef processing plants. The statistical process control parameters created for this study are a good initial estimation for the plant as few observations were observed outside the two boundaries parameters established in this study. However, it is recommended that a sound sampling plan is established to include more consecutive days (around 15 to 20) for robust decision making . AdditioFor small-sample process control charts, the first rule establishes that any observation falling above the +3σ limit should be considered as a point out of control ,23. ColdBio-mapping microbial indicators in this processing facility shows consistent reductions for all microorganisms evaluated up to the chilling location. Higher microbial loads were observed in all samples at pre- and post-evisceration with a continuous decrease as the process moved forward, until final interventions are applied. Then, a slight increase in counts occurred during the chilling process followed by a larger final increase after fabrication. The use of multi-hurdle interventions in the process can help with reductions in all indicator bacteria, which in turn can be translated in potentially lower levels of pathogenic bacteria. The variety of microbial loads obtained on different carcass regions throughout the process provides data to back up decisions on how to conduct microbial sampling based on worst case scenarios generated with data of their own process. The microbiological profile obtained in this facility not only provided a microbial baseline for supporting food safety continuous improvements to the plant, but also generated a comparative guideline for other beef processing facilities to evaluate the microbial control performance of their individual processes. The development of statistical process control parameters as a tool for food safety management to enhance the microbial performance on slaughtering and processing facilities resulted in basic reference values to the facility investigated to support their process improvements. However, more research and understanding of the process needs to be elucidated to interpret charts in applicable ways to evaluate the performance of food safety systems.


Group housing of preweaning dairy calves is increasing in popularity throughout the dairy industry. However, it can be more difficult to individually monitor calves to identify disease in these group systems. Automated milk feeders (AMF) not only provide producers with the opportunity to increase the milk allowance offered to preweaning calves but they can also monitor individual feeding behaviors that could identify calves at increased risk of disease. The objective of this retrospective case-control study was to determine how feeding behaviors change in preweaning calves leading up to and during a disease bout. This study was conducted between fall 2015 and fall 2016 on 2 commercial dairy farms in Ontario, Canada. Producers' treatment records for respiratory or enteric illness were used to identify cases. Control calves were selected from calves not treated for disease and matched on the days on the AMF. Both farms housed calves in dynamic groups of 9 to 11 calves with an AMF and fed milk replacer. Differences in feeding behaviors, including milk consumption, drinking speed, rewarded visits, unrewarded visits, and total visits to the AMF per day, were analyzed by mixed models accounting for repeated measures. Data were analyzed for the 7 d before, the day of, and 7 d after treatment. A total of 28 cases and 28 control calves (n = 56) were analyzed. Calves with disease consumed significantly less milk than their healthy counterparts, beginning 5 d before disease and until 3 d after disease detection. Sick calves had fewer unrewarded visits starting 3 d before until 2 d after illness detection. Sick calves drank significantly more slowly starting 4 d before illness detection until the day after illness detection compared with healthy controls. No differences were found between cases and controls for rewarded visits. Calves on a high plane of milk nutrition significantly alter feeding behaviors before illness detection. Data from AMF on feeding behaviors may help to detect disease earlier in preweaning dairy calves. Preweaning dairy calves are traditionally housed individually to decrease calf-to-calf transmission of pathogens . HoweverAMF). These computerized systems can decrease labor costs, allow producers to provide an elevated plane of milk nutrition . This study is reported following the STROBE-Vet reporting guidelines for observational studies . The datNCD) was treated with injections of an antibiotic  and an anti-inflammatory drug , whereas bovine respiratory disease (BRD) was treated with an antibiotic—either florfenicol combined with flunixin  or tulathromycin . On farm 2, NCD was treated with an oral antimicrobial  and BRD was treated predominantly with florfenicol  and meloxicam .During farm visits, birth records and treatment records for all calves were obtained. For the purpose of this study, treatment records were used as proxies for disease events. On both farms, producers used similar methods to identify sick calves. Both used a physical check to evaluate the attitude of the calf, an elevated body temperature (≥39.5°C), and potential signs of respiratory  or enteric disease. On farm 1, neonatal calf diarrhea  tag in their ear was scanned and individual behavioral feeding metrics were recorded onto a computer. For each case and control calf, all information pertaining to milk feeding plan for the preweaning period was obtained from the AMF to aid in analyses. These data included type of milk fed to the calves (milk replacer), milk feeding regimen , weaning method ], and age when fully weaned. Individual feeding metrics for each case and control calf were exported from the AMF Calf Manager software for the 14 d surrounding a treatment event. Feeding metrics included daily milk consumption [amount of milk consumed by each calf per 24-h period (L/d)], drinking speed [the average speed at which a calf ingested all of its milk meals in a 24-h period (mL/min)], rewarded visits , unrewarded visits , and total visits to the feeder (number of rewarded and unrewarded visits added together).As a calf entered the AMF, a radiofrequency identification  by day to determine the effect between health status and day relative to treatment. Calf was used as a random effect to account for repeated measurement in each model. Model fit was assessed for each model using residuals. The assumptions of normality and homoscedasticity of best linear unbiased predictors (BLUPs) were assessed graphically for each model. All models except unrewarded visits met all required assumptions of model fit. Attempts were made to normalize the unrewarded visits model through logarithmic and quadratic transformations but neither improved the fit, so the data were analyzed nonparametrically using a nonparametric kernel regression model. To determine sensitivity and specificity of each feeding behavior on disease prediction, optimal cut-points were determined separately for each variable using Youden's index.Calf treatment records and AMF data were imported into Excel (Microsoft Corp.) and cleaned and sorted. Excel files were then imported into STATA 15  for analysis. Descriptive statistics were reviewed for all outcome variables. Mixed linear regression models accounting for repeated measures with an unstructured correlation structure were built to evaluate the association between case or control classification and each of the following outcomes: total daily milk consumption (L/d), average daily drinking speed (mL/min), rewarded visits (visits/d), unrewarded visits (visits/d), and total visits (visits/d). Case/control status and day relative to treatment were forced as fixed effects, and an interaction term between case/control status and day was also evaluated. When the interaction term had A total of 28 cases and 28 controls  were enrolled in the study. On treatment day (d 0), healthy (control) calves were a mean (±SD) of 29 ± 11 d of age, and sick (case) calves were 25 ± 11 d of age. Farm 1 provided 19 cases  and 19 controls. Farm 2 provided 9 cases  and 9 controls. Healthy calves that were utilized more than once as a control were removed from the analysis. On both farms, herd-level disease prevalence was calculated using producer treatment records  for calves born and kept during the trial period . On farm 1, a total of 53 heifer calves were born over the trial period; of those calves, 27 were never treated, 2 were treated for both respiratory and enteric disease, 13 were treated for enteric disease, and 11 were treated for respiratory disease. This led to a disease prevalence of 49.1% for farm 1. On farm 2, a total of 26 heifer calves were born during the trial period; of those calves, 8 were never treated, 1 calf was treated for both respiratory and enteric disease, 2 calves were treated for enteric disease, and 15 calves were treated for respiratory disease. This led to an on-farm disease prevalence of 34.6%.P < 0.001), and the interaction between health status and day was significant (P < 0.001). Sick calves (cases) drank less milk than controls on d 5, d 4 , d 3 , d 2 , and d 1  before producer treatment compared with control calves (P < 0.001) less than control calves , and the interaction term between health status and day was significant (P = 0.002). Sick calves drank more slowly than control calves on d 4 , d 3 , d 2 , and d 1  before producer treatment , d 5 , d 3 , d 2 , and d 1  before producer treatment, as well as on producer treatment day . No differences were found with respect to unrewarded visits on d 6 or d 4 before producer treatment  decreased milk consumption on the day of illness detection compared with healthy calves , whereasUnrewarded visits to the AMF have been shown to be a useful predictor in disease detection in preweaning dairy calves. Our results are consistent with One limitation of this study was that health status was determined using producer treatment records. This may not be as accurate as daily health scoring in detecting sick calves. It is known that producer records are not always reliable and accurate for disease detection, and therefore may not be the best diagnostic tool . Many prThe results of this retrospective case-control study provide further evidence that sick calves on an AMF alter several aspects of their feeding behaviors leading up to a disease event. We found that sick calves drank more slowly and consumed less milk up to 4 and 5 d, respectively, before initiation of treatment by the producer. Sick calves also had fewer unrewarded visits to the AMF 3 d before illness detection by producer. However, our study was limited by the use of producer health records, which are less accurate than systematic clinical health scoring.


FL) to assess the shelf-life, while the lipid oxidation was measured by thiobarbituric reactive substance (TBAR) determination. The antimicrobial activity was assayed by means of classical methods and predictive microbiology. The experimental addition of polyphenolic extract led to 62% lower lipid oxidation and 58% higher antioxidant capacity; it also successfully modulated spoilage microbial populations with an average growth reduction of 15% on day 7. Results indicate that olive mill wastewater polyphenolic extracts could be added to raw ground beef meat to act as natural antioxidants and to modulate microbial growth.The mincing process of raw meat favors microbial spoilage as well as chemical and enzymatic oxidation processes. In order to limit this degradative process, preservatives are routinely added to minced meat products. The role of olive mill wastewater polyphenolic extract as a replacement for synthetic preservatives in beef burger was assessed. The antioxidant capacity of the extract experimentally added to beef burger was evaluated using the oxygen radical absorbance capacity method (ORAC Burger is one of the most appreciated meat products worldwide for its ease of preparation and versatility of consumption, which is considered a time-saving strategy in the modern lifestyle ,2,3. NevOleaceae family, that have been proven to inhibit or delay the rate of growth of a wide range of Gram-positive and Gram-negative bacteria as well as to have high antioxidant properties [Several spices, essential oils, extracts, powders and other plant by-products have been studied in recent decades in order to assess their activity and their effects on meat products as food supplementation ,17,18,19operties ,22. In poperties .Although the chemical composition and the antioxidant capacity of olive oil by-products as well as their application in foodstuff have been previously studied by several authors ,24,25, iIn this sense, the role of this natural extract in the replacement of synthetic preservatives in meat products is postulated. With this aim, the antioxidant activity and antimicrobial capacity of olive mill wastewater polyphenolic extract during beef burger shelf-life was evaluated. In order to estimate the potential activity of this polyphenolic extract in comparison with synthetic additives, different formulations were tested and compared to a control formulation and a control formulation with synthetic preservatives (sodium ascorbate) during a period of 7 days of cold storage.The crude phenolic extract (PE) used in the beef burger formulation was obtained through a three-step membrane filtration process using fresh olive mill wastewaters from processing olives of the Umbrian Moraiolo cultivar, as reported by Ianni et al. . To obtaBeef burgers were produced in an EU-approved meat processing plant located in Umbria, Central Italy. The meat for the preparation of beef burger was obtained from cuts (beef rump and shoulder muscle) of 18-month-old female Chianina cattle reared and slaughtered in Italy in accordance with European Union Regulation (Regulation (EC) No. 853/2004 s.m.i.) . After 1FL). One gram each of CM, PE and burger samples was separately mixed with a buffer, 75 mM, pH 7.2, containing 13.19 g of K2HPO4 and 10.26 g of KH2PO4 in 900 mL of deionized water, homogenized with an Ultra-Turrax homogenizer  for 1 min, and then vortexed for 2 min. The homogenates were centrifuged at 6000 rpm at 4 °C for 20 min, and the supernatant was used for the determination of the antioxidant capacity using the oxygen radical absorbance capacity method (ORACFL) based on the fluorescence decay rate of a probe in the presence of a radical oxygen species (ROO) and compared with that of a reference standard, Trolox . The ORACFL assays were carried out on a FLUO-star OPTIMA microplate fluorescence reader  at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The results are expressed as µg of Trolox equivalents (TE) per 100 g sample.The antioxidant capacity was evaluated for the synthetic and natural additives and burgers over the course of their shelf-life using the oxygen radical absorbance capacity method (ORAC2 = 0.9999), corresponding to a range of 0.4–4 mg of malonaldehyde (MDA)/kg meat. The MDA recovery was determined by spiking the samples with a known volume of 0.2 mM TMP. The TBAR concentration was expressed as mg MDA/kg meat.Lipid oxidation over the course of shelf-life was measured by thiobarbituric reactive substance (TBAR) determination, performed according to Tarladgis et al.  and measStaphylococcus aureus (WDCM 00034), Escherichia coli (WDCM 00013) and Pseudomonas fluorescens (WDCM 00115). The selected reference strains were revitalized in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 h with the exception of P. fluorescens, which was incubated at 25 °C for 24–48 h. An initial suspension of 0.5 McFarland in 0.9% sterile saline solution was prepared for each micro-organism, and 100 µL was then distributed on Mueller–Hinton Agar  plates with a swab, making four 90° rotations. At the time of use, the extracts were suspended with sterile demineralized water to obtain a concentration of 750 mg/mL, and then two dilutions were performed at 375 and 187 mg/mL. In each inoculated MHA plate, 7 mm-diameter holes were produced with a sterilized cork borer and then filled with 50 µL of extract suspension at different concentrations [The evaluation of the antimicrobial activity of CM and PE extract was performed through the agar well diffusion technique ,32 on sotrations ,32. The Enterobacteriaceae were enumerated according to a validated alternative method of ISO 21528-2 [Pseudomonas spp. count was performed on Pseudomonas Agar Base with CFC selective agar supplement (Oxoid Ltd.) and incubated for 48 h at 25 °C. Coagulase-positive staphylococci were enumerated on Baird Parker Agar with the addition of RPF supplement  and incubated at 37 °C for 48 h. Results were recorded as colony-forming units (CFUs) and converted into log10 values to obtain Log CFU/g of meat prior to statistical analysis according to Gill and Jones [Salmonella spp. detection was performed according to ISO 6579–1: 2017 [After 0, 2, 5 and 7 days of storage , 10 g of each sample was aseptically removed and placed in a sterile stomacher bag with 90 mL of Buffered Peptone Water . After homogenization , decimal serial dilutions were performed, and the below-reported microbiological determination was carried out in duplicate. Total viable count (TVC) was performed on Plate Count Agar  incubated at 30 °C for 72 h according to ISO 4833-1 [ 21528-2  , the enumeration of SO 16649  on all eSO 16649 .p < 0.05. The effects of formulation on the growth of the target micro-organisms were evaluated with the DMFit tool of the free predictive microbiology software Combase , allowing for the definition of growth parameters such as lag phase duration  and maximum growth rate  by means of the Baranyi and Roberts model [p < 0.05).Data were analyzed using the GLM procedure of SAS . An ANOVts model . The fitFL values are reported in A higher antioxidant activity was found in the PE in comparison with the CM containing ascorbic acid . The high antioxidant activity recorded in the PE is in agreement with a previous studies that reported the powerful antioxidant activity of olive phenolic compounds ,42,43,44S in the second part of storage (days 5–7) with a reduction of 62% of lipidic oxidation on day 7 for P compared to C samples. Similar results, albeit with a lower magnitude, have been reported by Martínez-Zamora et al. [Concerning lipid oxidation, increased TBARs values were detected during storage for all experimental formulations, and, among groups, differences were reported starting from 2 days with higher values for C samples. The addition of 700 mg/kg PE in meat (P group) guaranteed the lowest level of TBARa et al. , demonsta et al. .FL assay revealed differences in the antioxidant activity of four experimental groups, with the highest mean values recorded in the P group  increase was observed for all microbial populations and for all experimental groups studied as storage time elapsed. Immediately after production (T0), higher microbial populations were recorded for the TVC and LAB count followed by Pseudomonas spp. The initial (T0) TVC in all studied groups was approximately 4.4 Log CFU/g, which can be considered a characteristic value for minced meat products after manufacturing [Enterobacteriaceae counts  confirms the optimal initial microbiological quality attributable to the good physiological status of the animal at slaughter and to proper postmortem meat acidification as well as to the good hygienic conditions during slaughter, handling and production processes [The results of microflora evolution during storage for refrigerated beef burgers are depicted in acturing . Indeed,acturing ,49, albeacturing  or loweracturing . It has rocesses ,52.Pseudomonas spp. where the two microbial population with the highest initial value (T0), with average levels among experimental groups of 4.09 and 4.05 Log CFU/g, respectively. Similar counts were recorded for analogous products by Zamuz et al. [Pseudomonas in minced beef meat products.As above mentioned, following TVC, LAB and z et al.  and Andrz et al. , while sz et al.  and Marrz et al.  for Pseup < 0.001) differences between experimental groups were observed for TVC, Pseudomonas and Enterobatteriaceae (At the end of the storage period (day 7), significant . Considering Pseudomonas spp., the P group recorded a longer λ compared to C and A samples while AP was not statistically different from the other experimental groups; concerning μmax, both AP and P showed lower values compared to C and A. For this microbial population, the final value was significantly reduced in the P group.Regarding the TVC, the addition of polyphenols in beef burger resulted in an extended lag phase (λ) in P samples in comparison with C and A and a reduction in the maximum growth rate (μmax) in AP compared with all other groups and the final value for P. For Enterobacteriaceae, data show a reduction in the final value in the AP and P, while no significant differences were highlighted for λ and μmax; however, an increasing trend was recorded for λ and a decreasing one was noted for μmax .Neither λ nor the final value of the LAB population were different among groups, while the μmax was slightly higher in AP and P compared to C and A, agreeing with previous results from Servili et al. , who repP. fluorescens and Enterobacteriaceae and in a reduced μmax for P. fluorescens.In agreement with what is reported above, Mexis et al.  noticed As shown, the result of growth data modeling appears to be related to the influence exerted by the experimental addition of olive-mill-wastewater-derived polyphenols on extending the λ and reducing the μmax and the final value. Analogous conclusions have been previously reported for similar compounds and for other preservation methods, albeit a systematic comparison is difficult as the characteristics of microbial growth curves can be affected by differences in food matrices, storage condition and duration ,59.Concerning the antimicrobial activity, it has been demonstrated that the dialdehydic structure of olive phenols exerts an antimicrobial effect by strongly interacting with amino acids, proteins and membrane molecules, promoting membrane permeabilization and bacterial cell lysis . Indeed,Consumers’ interest in meat products formulated with natural preservatives has motivated researchers to evaluate the effectiveness and applicability of naturally occurring compounds with antioxidant and antimicrobial purposes. This study has proved that olive mill wastewater extracts are characterized by a phenolic profile and are able to significantly improve the oxidative and microbial stability of beef burger during cold storage.Therefore, it was concluded that olive mill wastewater extracts could be successfully added to raw ground beef meat to act as natural antioxidants and antimicrobials with added health, environmental and economic benefits as well as increased consumer appeal.


The highest values of moisture content, total soluble solids (TSS), pH, total phenolic contents (TPC), DPPH radical scavenging activity, yellowness (b*), and hardness were found in coated samples, which also showed the lowest values of the lightness (L*), greenness (a*), total viable count (TVC), yeast and mold counts, and acidity (p ≤ 0.05). Uncoated cucumber samples showed the highest (p ≤ 0.05) levels of acidity, lightness, greenness, TVC, and yeast and mold count. During storage, concomitant (p ≤ 0.05) reduction in moisture, TSS, pH, TPC, DPPH radical scavenging activity, L*, a*, b*, and hardness along with concurrent (p ≤ 0.05) increment in acidity, TVC, and yeast and mold count were evident in all cucumber samples. Interestingly, the changes in the aforementioned attributes were minimal in functionally coated samples in comparison to uncoated ones, suggesting the potential of OCE and OPE to preserve quality attributes of cucumber during cold storage.This study investigated the effect of functional coating using 2% chitosan and different concentrations of olive cake extract (OCE) and orange peel extract (OPE) on the physicochemical quality attributes of cucumber during cold storage at 4 °C for 21 days. Both coating and storage influenced ( Cucumis sativus L.) is one of the most important commercial vegetable crops cultivated in all regions worldwide with an annual production of 91.3 million tons in 2020 [Cucumber . However, prolonged storage significantly (p ≤ 0.05) reduced the moisture content to the minimum values at day 21 of storage of all samples. Improved moisture retention of coated cucumber samples is likely because the chitosan coating with or without functional ingredients acts as barrier against water evaporation, suppressing transpiration [The results on the moisture content, water activity (aw), and total soluble solids (TSS) of cucumber as affected by coating and storage are shown in piration . The redpiration . In agrepiration . In addipiration ,13,14,15The water activity of the cucumber samples was not influenced by both coating treatments and storage duration; however, it showed insignificant reduction during cold storage. The water activity of all cucumber samples stored at 4 °C was above 0.960, suggesting the susceptibility of cucumber to spoilage by mesophilic microbes if not well preserved. The decline of water activity during storage could be due to the reduction in moisture during the cold storage of cucumber. It is well known that fresh cucumber is susceptible to decay and spoilage by microorganisms, as it has a high moisture content (95%) and water activity (0.96) and consequently requires special processing treatments and preservation conditions .p ≤ 0.05) in cucumber coated with different concentrations of OCE or OPE, compared to the untreated control or samples coated with 2% chitosan only. The lowest TSS value was seen in untreated control samples stored for 21 days, whereas the highest value of TSS was observed in fresh cucumber samples treated with OCE and OPE, suggesting that functional ingredients in OCE and OPE improved the TSS of cucumber samples. The increased TSS in functionally coated cucumber could be due to the presence to reducing sugars in OCE and OPE used in the coating process of cucumber. In addition, coating cucumber with chitosan containing OCE and OPE might reduce the respiration rate, thereby maintaining higher TSS compared to uncoated samples. Similarly, previous reports indicated that cucumber coated with chitosan containing salicylic acid [p ≤ 0.05) in TSS was observed as the storage time increased to 21 days, reaching the minimum values at the end of storage of all samples. The reduction in TSS during cold storage is likely due to the senescence of the cucumber samples [The TSS increased significantly (lic acid  and carblic acid  as funct samples .p ≤ 0.05). Coating generally increased the pH of fresh and stored cucumber samples, and the highest values were observed in sample F that was coated with 2% chitosan containing 2% OPE, whereas the lowest values were observed in untreated control sample (A) during cold storage (p ≤ 0.05). Similarly, high pH values were observed in cucumber coated with modified corn starch/gelatin films [The results on the pH, acidity, total phenolic content (TPC), and antiradical activity . Generally, functional coating reduced the acidity of the cucumber samples, and the minimum values were seen in the samples coated with 2% OPE which remained the lowest during the storage of all samples, whereas the highest values were observed in the uncoated samples (p ≤ 0.05). The reduction in acidity following the coating treatment is likely due to the utilization of organic acids in respiration processes. During storage, the acidity of the cucumber samples was concomitantly (p ≤ 0.05) increased as the storage time elongated, and the highest values were recorded for samples stored for 21 days at 4 °C; however, the lowest increasing trend was seen in the coated cucumber samples. The increase in acidity during storage could be attributed to the conversion of sugars into organic acids [The acidity of the cucumber samples was affected by the coating treatment and storage duration p ≤ 0.0. Generalic acids . Previouic acids ,18,27.p ≤ 0.05). The increase in TPC of cucumber samples coated with functional ingredients (OCE and OPE) is likely due to the richness of these natural byproducts (olive cake and orange peel) with phenolic compounds [p ≤ 0.05) reduction as the storage period elongated, reaching the minimum values at the end of storage (21 days). In spite of the reduction in TPC during storage, its levels in uncoated cucumber samples remained the lowest, whereas those of the coated samples containing (2% OPE) remained the highest at all storage intervals (p ≤ 0.05). The reduction in TPC during storage is likely due to the utilization of these important phytochemicals as preservatives and antioxidant agents against microbial growth and oxidation of cucumber constituents. Hence, coating cucumber with functional ingredients from olive cake and orange peel could significantly improve the storage stability of this perishable product. The findings of this study are comparable to those of previous reports, which indicated that coated cucumber possessed higher total phenolic contents than uncoated samples, and it was reduced in a lesser manner during storage, compared to that of the uncoated samples [As shown in ompounds ,21. Duri samples ,27.p ≤ 0.05), indicating that the functional ingredients used in coating cucumber samples improved the functional properties of cucumber samples [p ≤ 0.05). It is worth noting that the DPPH radical scavenging activity of the cucumber sample coated with 2% OPE was 2 times higher than that of the untreated control at all the storage intervals, suggesting the antioxidative potentials of this functional material in coated cucumber samples, and hence could be used to enhance the nutritional and health quality and to extend the shelf life of cucumber [In accordance with TPC, the DPPH antiradical activity of uncoated cucumber was lower than that of samples coated with chitosan containing different concentrations (1% and 2%) of OCE or OPE . The hig samples ,27. Duricucumber ,27. In acucumber ,27,28,29p ≤ 0.05) increased to the maximum values at the end of storage. Cucumber samples coated with 2% OCE and 2% OPE showed the lowest counts, whereas the uncoated samples showed the highest counts at all storage intervals indicating the protective effects of the used coating materials during cold storage (4 °C). A food product carrying less than 6 log CFU is generally considered safe, and the results presented in p ≤ 0.05). The lightness values of uncoated cucumber were the highest at all storage intervals, which were reduced to the minimum values in cucumber coated with 1% OCE and then increased again for the samples coated with 2% OPE. The reduced lightness of coated cucumber could be attributed to the opacity and dark color of the functional coating materials [aterials . During aterials ,27. In aaterials ,27.p ≤ 0.05). In fresh cucumber, the greenness of uncoated samples was higher than those of coated ones, and greenness values generally reduced upon the coating of cucumbers with different concentrations of OCE and OPE. The reduction in greenness of cucumber following coating with functional materials could be attributed to the dark color of the added ingredients. Compared to fresh samples, prolong storage (21 days) greatly reduced the greenness values of cucumbers; however, the changes in greenness color were minimal in coated samples, suggesting the green color stabilizing effects of coating materials used in this study. The reduction in green color during storage could be due to the enzymatic decomposition of chlorophyll. The coating maintains the green color of cucumber by changing the surface environment of the samples, thereby preventing oxidative and enzymatic browning [The greenness (a*) values of cucumber were influenced by both coating treatment and storage duration in different magnitudes . In stored cucumber, the highest yellowness values were observed in cucumber coated with OPE, whereas the lowest values were seen in uncoated samples. The increase in yellowness of OPE-coated cucumber could be due to the yellow color of orange peel extract that might be released by microorganisms, thereby increasing the surface yellowness of the coated cucumber during storage. Similar changes in yellowness values of coated and uncoated fruits during storage were also reported [The yellowness (b*) values of cucumber were affected by both functional coating and storage duration in fluctuated ways. In fresh cucumber, the highest yellowness value was seen in the uncoated sample, which reduced to the minimum in samples coated with 1% OCE p ≤ 0.0. In storreported ,13. Overreported .p ≤ 0.05). The hardness of all samples concomitantly (p ≤ 0.05) decreased following the increase in storage time, reaching minimum values at the end of the storage; however, functionally coated cucumber exhibited higher hardness than uncoated ones, suggesting that functional coating delayed the decay of cucumber during prolonged storage at 4 °C. Generally, coating alleviates the negative effects of storage on the texture of cucumber due to the fact that coating materials form a barrier which reduces the O2 availability and metabolic rate, thereby delaying the softening process of coated samples during prolonged storage [The changes in the texture profile of cucumber as affected by functional coating and cold storage are shown in  storage . Hardnes storage . In agre storage ,13,15. T storage ,32. HencChemometric analysis was conducted by using the principal component analysis (PCA) and hierarchical clustering analysis (HCA) to intensely determine the combined effect of coating with different concentrations of orange peel extract (OPE) and olive cake extract (OCE) and storage time on the physicochemical properties of cucumber . The PCAFresh cucumber samples were obtained from a farm in Riyadh province during the harvesting season (March and April 2022). The samples were transferred to the Laboratory of Food Technology at the Department of Food Science and Nutrition, King Saud University under cooling conditions. Soon after arrival to the Laboratory, the samples were carefully checked, and damaged cucumber and foreign materials were removed. Cucumber samples of regular shape and same size were selected and washed using distilled water to remove dust. Kitchen paper towels were used to gently wipe off water from cucumber surfaces prior to coating experiments. Fresh orange was obtained from a local farm and peeled manually to obtain fresh orange peels, which were dried oven at 45 °C until obtaining constant weight . Fresh olives were acquired from a local market, and the cold press technique was used for oil extraction from the samples using a SUS 304 oil extractor . The olive cake was dried (moisture content 11.32 ± 1.43%). Both the dried olive cake and orange peel were ground into fine powders and kept in sealed bags at 4 °C for further use. All chemicals used were of analytical grade and were obtained from Sigma-Aldrich .2O at 70 °C. After that, the extract was cooled to room temperature, filtered using Whatman filter paper #1 thimbles , and the filtrates were collected. The extraction process of the residues was repeated thrice, and the filtrates were collected, combined and dried at 50 °C using a rotary evaporator. The obtained OCE and OPE were ground and stored in sealed bags at −20 °C for further use.The preparation of OCE and PCE was carried out as described previously . Brieflyw/v), thoroughly mixed, and then homogenized for 5 min using an Acapulco 30564 kitchen blender  until obtaining a smooth solution. The prepared chitosan solutions with or without functional ingredients were used for the coating of cucumber samples as described below.The chitosan solution containing functional ingredients was prepared freshly as described in our recent study . In brieFor functional coating, six samples (blocks) were prepared as follows: A , B (coated with chitosan (2%) only, positive control), C (coated with chitosan (2%) containing 1% OCE), D (coated with chitosan (2%) containing 2% OCE), E (coated with chitosan (2%) containing 1% OPE), and F (coated with chitosan (2%) containing 2% OPE). Fresh and cleaned cucumber samples were dipped into the above coating solutions for 5 min, carefully removed from the dipping solutions, and dried on the stand sieves for 2–3 h at room temperature with the help of a CK2215 plastic fan  for the quick and complete drying of coated cucumber samples. After that, cucumber samples were packed in perforated (6 holes on the lid) plastic containers and stored in the refrigerator (4 °C) for up to 21 days. The entire blocks were replicated three times and at 7-day intervals, the cucumber samples were analyzed for physicochemical quality attributes.The moisture content, total soluble solids, and treatable acidity were determined using the standard official method . The moiThe surface CIE color attributes  of the cucumber samples during storage were determined using a CR-300 Minolta colorimeter . The colorimeter was first calibrated using a white plate, and then three readings were taken from different areas of each sample. Texture profile analyses of cucumber samples at different storage intervals  were measured using a Brookfield CT3 texture analyzer  as described in the instruction manual. A two-cycle test was used for triplicate analyses of the textural attributes: hardness (kg), cohesiveness, and springiness (mm).The total viable, mold, and yeast counts were determined using pour plate methods on plate count agar and potato dextrose agar, respectively, as described previously . BrieflyPrior to analysis of the TPC and DPPH radical scavenging activity, a water extract of the cucumber samples was prepared by homogenizing 10 g of the sample in 100 mL distilled water, followed by filtration and centrifugation to obtain clear extract. For analysis of TPC, the Folin-Ciocalteu’s (FC) method was used as described previously . Brieflyp ≤ 0.05 and data were presented as means ± standard deviation (means ± SD) of three measurements. MULTBIPLOT software was used for the analysis of principal component analysis (PCA) and hierarchical cluster analysis (HCA) of data variables of the samples during storage intervals as described by Vicente-Villardón [The experiments were designed using a completely randomized block design with six treatments , and measurements of the physicochemical quality attributes were taken at intervals of 0, 7, 14, and 21 days of cold storage. The entire blocks were independently replicated three times on three different days and the measurements of each quality attribute were taken in triplicate. The data obtained were subjected to analysis of variance (Two-way ANOVA) using SAS software , and the means were compared using Duncan’s multiple range tests (DMRT) and the general linear model (GLM). Significance was accepted at illardón .This study concludes that coating treatment and cold storage at 4 °C influenced the physicochemical, nutritional, and health quality attributes of cucumber at varied magnitudes. A functional coating with different concentrations of olive cake extract (OCE) and orange peel extract (OPE) increased the total soluble solids, hardness, total phenolic contents (TPC), and DPPH radical scavenging activity of the cucumber samples. However, prolonged storage (21 days) reduced total soluble solids, hardness, TPC, DPPH inhibition, and moisture content, and increased the total viable count (TVC), yeast and mold counts, and acidity of the product. Interestingly, functional coating alleviates the negative effect of storage on the above quality attributes. Overall, functional coating with OCE and OPE improved the physicochemical quality attributes and may extend the shelf-life of cucumber for up to 14 days of refrigerated storage.


Hearing loss was proposed as a factor affecting development of cognitive impairment in elderly. Deficits cannot be explained primarily by dysfunctional neuronal networks within the central auditory system. We here tested the impact of hearing loss in adult rats on motor, social, and cognitive function. Furthermore, potential changes in the neuronal activity in the medial prefrontal cortex (mPFC) and the inferior colliculus (IC) were evaluated.In adult male Sprague Dawley rats hearing loss was induced under general anesthesia with intracochlear injection of neomycin. Sham-operated and naive rats served as controls. Postsurgical acoustically evoked auditory brainstem response (ABR)-measurements verified hearing loss after intracochlear neomycin-injection, respectively, intact hearing in sham-operated and naive controls. In intervals of 8 weeks and up to 12 months after surgery rats were tested for locomotor activity (open field) and coordination (Rotarod), for social interaction and preference, and for learning and memory . In a final setting, electrophysiological recordings were performed in the mPFC and the IC.P < 0.05). Learning the concept of the radial maze test was initially disturbed in deaf rats (P < 0.05), whereas retesting every 8 weeks did not show long-term memory deficits. Social interaction and preference was also not affected by hearing loss. Final electrophysiological recordings in anesthetized rats revealed reduced firing rates, enhanced irregular firing, and reduced oscillatory theta band activity (4–8 Hz) in the mPFC of deaf rats as compared to controls (P < 0.05). In the IC, reduced oscillatory theta (4–8 Hz) and gamma (30–100 Hz) band activity was found in deaf rats (P < 0.05).Locomotor activity did not differ between deaf and control rats, whereas motor coordination on the Rotarod was disturbed in deaf rats (Minor and transient behavioral deficits do not confirm direct impact of long-term hearing loss on cognitive function in rats. However, the altered neuronal activities in the mPFC and IC after hearing loss indicate effects on neuronal networks in and outside the central auditory system with potential consequences on cognitive function. Hearing loss is one of the most frequent sensory deficits affecting more than 5% of the worldwide population . In eldeAnimal models offer the opportunity to investigate the causality between hearing loss and cognitive decline in a confined setting, especially in the absence of language, which may act as a confounding factor. Numerous studies investigated the effect of hearing loss on auditory function and neural plasticity throughout the auditory pathway . The impThe PFC is a key area for the regulation of higher brain functions. It is involved in various cognitive processes with higher-level stages of information processing, as needed in the categorization and recognition of prosodic stimuli . In ordeWe here aimed to examine a possible causal relationship between hearing loss and cognitive impairments in an animal model with defined hearing loss, and to identify possible neural correlates within the auditory system and the PFC. Hearing loss was induced with intracochlear injection of neomycin in adult rats. Thereafter, all rats were regularly tested for social behavior and motor function up to 1 year after deafening. Cognitive behavior after hearing loss was tested in the 4-arms baited 8-arms radial maze test, starting 8 weeks after surgery to allow plastic neuronal changes after deafening to occur. With this paradigm different aspects of cognitive behavior can be addressed (a) procedural memory, i.e., knowledge that some of the arms are baited and that food can be obtained from a baited arm only once during each training trial. In order to reach a certain criterion of success , rats usually need to be trained for 3–4 weeks with 6 runs per day. At this point, (b) reference memory, i.e., remembering, which arms had been baited before, can be challenged by introducing days or even weeks between testing. Additionally, once the rats have learned the paradigm, cognitive flexibility (c) can be tested by baiting arms that have never been baited for a certain rat before, which is called “reversal learning”. One year after hearing loss, rats underwent electrophysiological recordings of single unit activity (SUA) and local field potentials (LFP) in the medial PFC (mPFC) and the IC of the central auditory pathway in a final setting. Within the mPFC, electrophysiological recordings were acquired at the prelimbic area, since this subarea is suggested to be involved in information processing for working memory and for spatial memory, as well as for the selection and maintenance of learning strategies . Furthern = 26) were housed in groups of two to four animals per Makrolon Type IV open cages under controlled environmental conditions . Tap water was freely available and standard rodent chow  was fed with 14–16 g per day and animal to enhance saliency of reward pellets while ensuring weekly weight gain of about 5–15%. Body weight and clinical scores were assessed at least two times a week to ensure the well-being of the rats.Adult male Sprague-Dawley rats . All efforts were made to minimize the number of animals and their suffering.n = 12) where hearing loss was induced via intracochlear injection of neomycin, (2) sham group (n = 5) where the same surgical procedure was performed without opening the inner ear and without injection of neomycin, and (3) naive group (n = 9) which served as controls. Only rats of one experimental group were housed together. Acoustically evoked brainstem responses (ABR) measurements were conducted before surgery in all rats to ensure normal hearing; after surgery successful induction of bilateral hearing-loss was confirmed in the deaf group, respectively, normal hearing in the sham and naïve group by ABR measurements. At the end of the study, the hearing status of each animal was verified in all three groups with final ABR measurements. When first placed into the open field, exploratory behavior will play a major role for outcome and will also affect first time testing of social interaction, whereas during repeated testing rats already remember the open field. With regard to Rotarod testing, animals improve especially during first time exposure since animals learn to maintain balance (At age of 8–9 weeks (mean weight of 240 g), animals were randomly assigned to one of the three experimental groups: (1) deaf group , complemented by infiltration of the postauricular region with local anesthetic  and systemic analgesia . Rats were placed in a lateral recumbent position, the skin incised for an approximate length of 3 cm, the muscles pulled caudally and the bulla exposed. A slow speed 0.5 mm diameter diamond burr was used to drill a small hole into the bulla to display the round window, the tympanic cavity, and the ossicles. For the administration of neomycin via the round window, 5 mg neomycin was dissolved in 1 ml of PBS (adjusted to pH 7.4). A lancet was used to perforate the round window and a 30-gauge needle and micro syringe (Hamilton) were used to inject 15 μl neomycin . The bulABR were recorded in all rats under general chloral hydrate anesthesia directly before and after the surgery as well as before the final electrophysiological measurements. ABR measurements were performed using subdermal needle electrodes, which were positioned at the vertex of the skull, behind the left or right ear and in the neck as grounding electrode. ABRs were obtained from vertex vs. retroauricular electrode by 40 dB preamplification  and 60 dB amplification . A second order bandpass filter  was used to eliminate low-frequency signals from the recordings.Stimuli were generated by Audiology Lab stimulus generation and acquisition system  running on a PC and connected to an AD converter  and an analog USB-controlled attenuator  connected to a calibrated DT48 speaker  positioned close to the ear. The clicks of 50 μs duration were presented at levels from 10 to 100 dB SPL p.e. in 10 dB steps in control and sham rats. After intracochlear injection of neomycine, however, rats were tested for ABR up to 120 dB to ensure complete hearing loss. The interstimulus interval was 113 ms and clicks were repeated 200 times for each stimulus level. The ABR signals were recorded using a hardware filter . For analysis, the recorded ABR signals were additionally digitally filtered  and averaged using custom codes in MATLAB . ABRs were determined manually by detection of clearly identifiable waves II and III at latencies between 2 and 4 ms in ABR signal .All rats were tested just before surgery (Pre-surgery), in weeks 1, 2, 4, 8, 16, 24, 32, 40, 48, and 56 after surgery. Behavioral testing of motor activity and interaction was done in a separate room with dim lighting. Two rats were first tested for motor activity in two open fields, thereafter placed into a third open filed for social interaction. Then, rats were tested consecutively on the Rotarod in another room with dim light.Locomotive behavior was tested in an open field arena (60 cm × 60 cm × 30 cm). When first placed into the open field, exploratory behavior will play a major role for outcome whereas during repeated testing rats already remember the open field. Therefore, 1 week before testing started, rats were accustomed to the arena. For actual testing, rats were placed in the open field for 10 mins. The distance was automatically evaluated using video recordings from above the arena .The effect of hearing loss on motor coordination and balance was assessed by placing the animals on a rotating rod inside a test chamber . The rod rotated with accelerating speed for 60 s  followed by another 60 s with constant speed (15 rotations per minute). One week before testing started, rats were trained to stay on the Rotarod. For actual testing, rats were placed on the rod for three consecutive trials and time was measured until the rat dropped of the rotating rod. For analysis, the mean latency of all three trials was calculated.To evaluate if hearing loss affects the social interaction, two rats  were placed into an empty open field arena for 10 mins. Social interactions were video recorded via a camera installed above the test arena. One week before testing started, rats were accustomed to being placed for social interaction in the open field. For analysis the duration of playing, following, anogenital, and frontal sniffing were evaluated. Total interaction duration was calculated as the sum of all evaluated behaviors.Here testing started 8 weeks after surgery to allow plastic changes to neuronal network activity after deafening/sham-deafening before being tested. Rats were once tested for social preference on the day before each 8-arm maze session. Thereafter, 8-arm maze testing session was done in a separate room with full light.Testing in this paradigm started 8 weeks after surgery. After an acclimatization phase of 5 mins an object stimulus (empty wire mash cage) was introduced to the test rat. After additional 4 mins, the object stimulus was replaced by an identical cage filled with a male, age-matched, unfamiliar social interaction partner for another 4 mins . The preTo investigate if the hearing loss has an effect on learning and memory, training in the radial 8-arm maze started 8 weeks after surgery. The maze consisted of eight arms (12 cm × 76 cm) equally distributed around a central platform (35 cm diameter) in 80 cm above floor level. At the far end of each arm a cup was installed that contained reward pellets . Training started with a habituation phase in which each animal was able to freely explore the whole maze.Testing in the 4-arms baited 8-arms radial maze paradigm can be divided into three sections: (a) training rats to collect reward pellets from certain arms of the maze until reaching a certain criterion of success, which usually needs training the rats with 6 runs per day for 3–4 weeks, (b) retesting rats every 8 weeks. For final reversal learning (c), the previously baited arms were now unbaited (and vice versa) to test rat’s cognitive flexibility.For training, four arms were randomly chosen for each individual rat (not more than two adjacent arms) and equipped with three reward pellets in each cup. Each run started by placing the animal facing a random direction in the center of the maze and was terminated if all rewards were collected by the animal or after 15 mins. On the first two training days the animals were tested with three consecutive runs (one block) and on the following days with six runs (two blocks). The sequence of entrances was tracked from outside the room via video recordings. For analysis, the number of entries in unrewarded arms [reference memory error (RME)] and the number of previously entered arms during the same run [working memory error (WME)] were assessed. The training was terminated upon “completion criterion” of less than three WME in three consecutive blocks of three runs, together with less than two RME per block.After initial training for completion criterion, we focused on long-term memory by retesting rats in the radial maze test every 8 weeks. Here, rats were tested with six runs per day for two consecutive days . Since some of the rats did not reach the initial training criterion, and consequently were disadvantaged at the following retesting, rats were retrained for criterion at the third (at week 32) and at the final retraining session (at week 56).As final test all animals were subjected to a reversed training phase in which the reward pellets were placed in the previously unrewarded arms. Here rats were tested with six runs per day for two consecutive days .For the extracellular SUA measurements and LFPs recording after final behavioral testing, rats were anesthetized with urethane ; with additional doses as needed, depth of anesthesia was checked by the foot pinch. Rats anesthetized with urethane demonstrate spontaneous and cyclical alternations of brain state that resemble sleep state alternations or the global active or awake state of the brain. These fluctuations between different brain states are known as rapid eye movement (REM) and non-REM stages . NotablyThe incision site was infiltrated with a local anesthetic, Xylocain. Rats were placed in a stereotaxic frame and body temperature was maintained at 37 ± 0.5°C by a heating device . Small craniotomies were made over the target coordinates for the mPFC and the IC. Thereafter the animal was placed in a Faraday cage to minimize electrical noise. A single microelectrode for extracellular recordings , diameter 80 μm, and impedance 1–2 MΩ) was connected to the Mini Matrix 2 channel version drives headstage . The microelectrode signal was passed through a headstage with unit gain and then split to separately extract the SUA and the LFP components. For SUA recording, signals were band pass filtered between 500 and 5,000 Hz and amplified from 9,500 to 19,000 and sampled at 25 kHz. The stainless steel guide cannula, which touches the cortical surface, served as reference for the microelectrode  served as electrode for recordings of the electrocorticograms (ECoGs). Additionally, a needle reference electrode was inserted in the neck muscles. The signal was filtered by using band pass (0.5–100 Hz) with a sampling rate of 1 kHz. Data were acquired using the CED 1401 A/D interface . After termination of the experiment, electrical lesions were made at the recording sites to allow histological verification of the localization  as previously described .Action potentials from single neurons were discriminated by the template-matching function of the spike-sorting software . Only well isolated SUs were included in the analysis, which was determined by the homogeneity of spike waveforms, the separation of the projections of spike waveforms onto principal components during spike sorting, and clear refractory periods in inter-spike interval (ISI) histograms. All analyses were performed using custom-written MATLAB  functions unless otherwise noted.2/mean ISI) to compare the variation in their median values. A higher random firing pattern would be expected to have an increase in the diversity of the ISI lengths and higher DI of FR. A lower DI would be indicative of less diversity of the ISI lengths and more regular activity. The asymmetry index (AI) of firing was obtained by determining the ratio of the mode to the mean ISI, which provides information on the shape of the ISI histogram or the regularity of the discharge pattern. An asymmetry index close to 1 reveals a relatively regular firing pattern, whereas the more the index decreases from 1 the more irregular are the spike trains.Firing rate (FR) was analyzed by taking the reciprocal value of the mean ISI for the whole 100 s of recording of spontaneous activity. The dispersion index (DI) of FR, a measure variance of firing rate was calculated by dividing the square of the standard deviation of the ISI by the mean ISI  were identified by visual inspection and excluded. The spectrograms were computed by the squared magnitude of the short-time Fourier transform. The Fourier transformation was performed in each epoch and then averaged across all epochs. A finite impulse response 50-Hz notch filter and a 100-Hz low-pass filter were used. The spectral power of mPFC- and IC-LFPs, and SMCtx-ECoGs were derived by discrete Fourier transformation with blocks of 1,024 samples using a Welch periodogram in a custom Matlab script, which resulted in a frequency resolution of 0.9766 Hz. Hanning’s (referred to as Hann) window function was applied to control spectral leakage phenomenons. The relative power indices for each band were calculated from the absolute power in each frequency band and expressed as a percentage. For comparison of powers at different frequency bands, the area under the computed power density spectrum in specified frequency ranges for theta (4–8 Hz), alpha (8–12 Hz), beta (12–30 Hz), and gamma (30–100 Hz) frequency bands were calculated and averaged.Further, the functional relationships between the SMCtx-ECoGs and LFPs of mPFC, and IC were estimated by means of coherence using methods described by After electrophysiological recording, animals were euthanized and transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in 0.1 m PBS. Brains were removed and postfixed in 4% paraformaldehyde followed by immersion in 30% sucrose for at least 24 h. Each brain was cut into 20-μm sections and stained with a standard hematoxylin and eosin (HE) protocol to verify the position of each electrode.For histological verification of hearing loss, cochleae were prepared. Immersing with Spalteholz solution  allowed to take representative images of one deaf and one naïve control rat with a Leica TCS SP8 confocal laser scanning microscope using a 10× -objective .F-test followed by post hoc Bonferroni t-test for multiple comparisons with the factors group and repeated testing was used. For the comparisons of the electrophysiological data between control and the deaf group in either mPFC, IC, or SMCtx, a Mann-Whitney U test was applied to test for significant differences. The characteristics of the ISI histograms  did not show normal distributions in most cases and therefore were analyzed with nonparametric multifactorial statistical methods. All tests were used two-sided; a P < 0.05 was considered significant.Rats were first randomly assigned to the deaf, sham, or naïve group. Thereafter, preoperative measures for locomotor activity in open field, Rotarod and social interaction were compared to ensure that groups did not unintentionally differ before surgery for deafening or sham-deafening. Statistical analyses were performed using SigmaStat 4.0 software . For behavioral testing, two-way repeated measure analysis of variance (ANOVA) n = 12 in the deaf group, n = 9 in the naïve group, and n = 5 in the sham group. After intracochlear neomycine injection, two animals were excluded from the deaf group because the ABR measurements showed insufficient success of the deafening surgery. These rats were entirely excluded from statistical analysis, leaving n = 10 rats as group size of the deaf rats. During the course of the experiments, two rats of the deaf group had to be sacrificed after testing in week 24, respectively, after testing in week 40 because of bad general condition not associated with the experiments, leaving n = 8 deaf rats for final behavioral testing. One animal of the sham group died during surgery, leaving a total of n = 13 animals for the control group . Moreover, one rat of the control group had to be sacrificed after testing in week 32, leaving n = 12 for final analysis.A total of 26 male normal hearing rats were used for the present experiments which were randomly assigned to F1,40 = 3668.7, P < 0.001; post-hoc testing, P < 0.05) and compared to the naïve and sham group , whereas directly after the intracochlear injection of neomycin and at the final measurement 56 weeks after surgery the thresholds of the ABR measurements in the deaf group were significantly elevated to at least 100 dB. This was significantly enhanced both compared to the pre-OP time point (two-way ANOVA factor group Representative cochlea were stained to show hair cell loss after neomycin injection . In naivn = 4) and naïve (n = 9) rats, both groups were combined as control group (n = 13) for further analysis. For analysis of open field, Rotarod, and social interaction by repeated measure ANOVA missing rat data were replaced by group mean. This affected one rat of the control group after week 32, as well as two rats of the deaf group after week 24, respectively, week 40 on).Since ABR and behavioral data did not differ between sham  did not differ between groups .F1,126 = 4.685, P = 0.042, or “filled”: F1,126 = 7.028, P = 0.015; F1,21 = 7.19, P = 0.014) as well as for the factor empty/filled , whereas the interaction between factors was not significant . Post-hoc testing showed that deaf rats interacted less with the empty and the filled cage than the control group (P < 0.05). Both groups preferred interaction with the filled cage as compared to the empty cage . For statistical analysis of all radial maze settings blocks of three runs were used. During initial training for performance in the 4-arms baited 8-arm radial maze, starting week 8 after surgery, deaf rats made more total errors during training than the control group. Although ANOVA only showed a trend for significance for the factor group , we nevertheless performed post-hoc group comparisons, which shows significantly enhanced errors in blocks 1, 2, and 7 . Also retraining for performance at weeks 32 and 56 revealed no differences between groups . Finally, deaf and control rats did not differ during reversal training  recorded per individual rat in the control group in the mPFC was 47.0 ± 5.4 and in the IC 32.83 ± 6.4. In the deaf group, the average recorded per individual rat for the mPFC was 65.8 ± 6.7 and for the IC 43.77 ± 9.26. An example of 30 s recording epochs of the extracellular single neuron spikes and its raster plots from the mPFC and the IC, together with action potential waveform of the mPFC and of the IC are shown in U test showed that the FR in the mPFC was lower in the deaf group compared to the control group (P < 0.001), while in the IC this difference did not reach a level of significance (P = 0.11). The DI in the mPFC was higher in rats from the deaf group compared to the control group (P < 0.001). The DI of IC neurons did not differ between the control group and the deaf group. Furthermore, the AI of mPFC and IC neurons did not differ between the control group and the deaf group (The Mann-Whitney af group .n = 176), the mPFC deaf group (n = 134), as well as from the IC control group (n = 128) and the IC deaf group (n = 128).For LFP and coherence analysis, we used neuron epochs from the mPFC control group . In the deaf group, alpha oscillatory activity was higher and beta oscillatory activity was lower only in the SMCtx-ECoG as compared to the control group (P < 0.05), while activity in mPFC and IC did not differ. For gamma oscillatory activity, analysis showed that the relative power was lower in rats from deaf group in the IC area , while gamma activity in the mPFC did not differ (The Mann-Whitney t differ .P < 0.01). For the IC and the SMCtx-ECoG the coherency for alpha (P < 0.05), beta (P < 0.01) and gamma (P < 0.01) was higher in the deaf group as compared to the control group (Analysis of the coherence between the spontaneous LFP signals recorded from mPFC and SMCtx-ECoG showed that the theta frequency band coherency was reduced in the deaf group, while the gamma frequency band coherency was enhanced as compared to control groups (ol group .We here investigated whether deafening by local intracochlear injection of neomycin in adult rats would have long-term effects on motor function, non-auditory cognition, social interaction, and neuronal activity in brain regions associated with higher cognitive function.Deaf rats had early learning deficits when training of the 4-arms baited 8-arms radial maze task started 2 months after surgery. Later during training, however, the number of working or reference memory errors did not differ between deaf rats and control groups. Re-testing every 2 months for up to 1 year, or final testing with reversal of baited arms also did not show differences between groups. These findings corroborate a recent study, where 30 days after noise-exposure rats with hearing deficits had difficulty in learning the initial rule of spatial paradigms, but only a tendency to not remember it during retesting . In miceTogether, while such data demonstrate effects of hearing loss on cognitive function and social interactions, the rodent data overall do not support the hypothesis that hearing loss by itself could induce a progressive cognitive decline (reviewed in In humans, it has been found that hearing loss increases the risk of social isolation and loneliness , which aAfter neomycin induced hearing loss, rats were impaired on the Rotarod, a test traditionally used to assess motor deficits in rodents . In humaDespite only minor effects on behavior tested in this study, we found clear long-term effects of deafening on neuronal activity in the mPFC, a core structure for higher-order cognitive and executive functions . The preIn the present study the firing rate of the IC was reduced 1 year after deafening, whereas different groups reported on hyperexcitablity in regions of the central auditory pathway that develop within the first few weeks after hearing loss until up to 6 months after the trauma , 2017. HThe auditory perception depends on the integration of multiple sensory and cognitive domains within the central auditory system as well as non-auditory regions. Nevertheless, the networks sub-serving this integration are unclear. Retrograde tracing studies in rats have shown that the IC of the auditory midbrain receives dense descending projections not only from the auditory cortex, but also from the visual, somatosensory, sensory, motor, and prefrontal cortices .Besides SUA, oscillations and rhythmic activities of functional neural networks are fundamental for complex perceptual and cognitive functions, including speech and social communications , i.e., aIn the IC, theta and gamma band oscillatory activity was reduced, whereas coherence of alpha, beta, and gamma between IC and SMCtx was enhanced 12 months after deafening. How altered oscillatory activity may affect processes that contribute to speech processing has not been investigated, yet .It has been described that altered neuronal activity within frontal cortical areas may serve as a compensatory mechanism for deficits in speech processing in older adults, particularly in more challenging listening conditions such as hearing in noise . AlthougThe original contributions presented in this study are included in the article/This animal study was reviewed and approved by Lower Saxony State Office for Consumer Protection and Food Safety.MJ, SH, PH, JK, VS, AK, and KS: study concept. MJ, SH, MA, JJ, and KS: laboratory work and analyses. MJ and KS: scientific writing. All authors carefully revised the manuscript and approved the submitted version.