The objectives of this study were to evaluate the impacts of two modified-live virus (MLV) vaccination protocols and respiratory disease (BRD) occurrence on the microbial community composition of the nasopharynx in feedlot cattle.n = 525) arrived in 5 truckload blocks and were stratified by body weight, sex, and presence of a pre-existing identification ear-tag. A total of 600 nasal swab samples were selected for DNA extraction and subsequent 16S rRNA gene sequencing to characterize the microbiome of the upper respiratory tract. Nasal swabs collected on d 28 from healthy cattle were used to evaluate the impact of vaccination on upper respiratory tract (URT) microbial communities.The treatment groups included in this randomized controlled trial included: 1) no viral respiratory vaccination (CON), 2) intranasal, trivalent, MLV respiratory vaccine in addition to a parenteral BVDV type I and II vaccine (INT), and 3) parenteral, pentavalent, MLV respiratory vaccination against the same agents (INJ). Calves  and this difference was attributed to decreased relative abundance (RA) of Mycoplasma spp. (P = 0.04). Mannheimia and Pasteurella had lower RA in INT (P < 0.05). The microbiome in healthy animals on d 28 had increased Proteobacteria (largely Moraxella spp.) and decreased Firmicutes  compared to animals that were treated for or died from BRD (P < 0.05). Cattle that died had a greater RA of Mycoplasma spp. in their respiratory microbiome on d 0 (P < 0.02). Richness was similar on d 0 and 28, but diversity increased for all animals on d 28 (P>0.05).Firmicutes were less abundant in INT calves ( Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni (Hs), are widely considered to be the most important causes of acute onset pneumonia  is the most common reason for antimicrobial drug treatment among feedlot cattle in North America  is unknown. In the feedlot setting, vaccines administered against viral respiratory agents contain modified-live virus (MLV) antigens that can be administered either intranasally or parenterally, with different immunological responses expected. Parenteral vaccination primarily induces a systemic immune response within the host  in affecting BRD occurrence . One vaccination group received an intranasal MLV respiratory virus vaccine containing bovine herpes virus type 1 (BHV1), BRSV, and parainfluenza virus type 3 (PIV3) and a parenteral vaccine containing bovine viral diarrhea virus (BVDV) types I and II (INT), and a group vaccinated parenterally against the same agents using a pentavalent MLV respiratory vaccine . Samples of respiratory secretions in the nasopharyngeal region were obtained on d 0 and d 28 and upon initial treatment for BRD. Samples were analyzed using 16S rRNA gene sequencing to characterize microbial community structures and determine differences among vaccine treatment groups, among cattle with different health outcomes, and over time. Administration and handling of vaccines followed label and Beef Quality Assurance guidelines. Animal use protocols were approved before the initiation of the study by the West Texas A&M University (WTAMU) Institutional Animal Care and Use Committee (IACUC #2020.10.002).n =12 per pen, 15 pens per treatment) , stratified by truckload (n = 5), body weight, sex (bull [n = 129] or steer [n = 396]), and the presence of a pre-existing identification ear tag. As such, these factors were homogenously represented across pens within truckload blocks and cattle housed in a pen belonged to the same vaccine treatment group. The INT vaccination group received a trivalent MLV respiratory vaccine administered intranasally  and a parenteral vaccine containing BVDV types I and II given subcutaneously . The INJ group received a parenteral, pentavalent MLV respiratory vaccine given subcutaneously . To minimize the potential transmission of infectious agents between treatment groups (including vaccine-origin viruses), an empty pen was maintained between treatment groups to eliminate direct contact. Additionally, initial processing, subsequent sample collection, and BRD treatments were conducted in the order of CON, INJ, and then INT treatment groups to minimize the potential for vaccine or natural virus transmission.A total of 525 crossbred beef bull and steer calves (213 ± 18.4 kg) were acquired from auction markets in central Texas and enrolled in the study. Cattle were randomly allocated to experimental treatment pens  and tildipirosin as a metaphylactic antimicrobial drug . Bulls were band castrated  and treated with an analgesic .This study was conducted from November 2020 to May 2021 at the WTAMU Research Feedlot, located near Canyon, TX. Upon feedlot arrival (d−1), individual body weight (BW), sex (bull or steer), and the presence of a pre-existing ear tag were recorded  to each animal. A CIS of 1 described a “normal” steer without any visible clinical signs of sickness. Steers with a CIS of 2 represented a “moderately ill” steer, which appeared gaunt, had nasal/ocular discharge, lagged behind others, and coughed. A CIS score of 3 indicated a “severely ill” steer with purulent nasal/ocular discharge, labored breathing, and severe depression. Lastly, CIS 4 corresponded to a “moribund” steer that was unresponsive to human approach and near death. Animals assigned as CIS of 2 or 3 were removed from the pen and further examined, including measurement of rectal temperatures. Steers assigned CIS of 3 or 4 were classified as BRD cases, as were animals assigned CIS of 2 if they also had a rectal temperature ≥39.7°C. Cattle classified as CIS 4 were euthanized. Because tildipirosin was administered metaphylactically on d 0, no other antimicrobial drug (AMD) treatments were administered until after d 7. After this period, BRD cases were treated with an AMD, using a standardized treatment protocol . Steers first diagnosed with BRD were treated with florfenicol . Following the expiration of a 3-day post-treatment interval (PTI), steers again meeting the same BRD case definition were treated with enrofloxacin . After another 3-day PTI, steers meeting the BRD case definition a third time were eligible for a third and final treatment with ceftiofur crystalline-free acid . Steers that received three antimicrobial treatments with < 0.45 kg ADG since d 0 or body condition score < 3 of 9 were removed from the study and considered chronically ill. Cattle that died or were euthanized underwent post-mortem examination, and the cause of death was recorded. Further information about the study population, cattle handling, and the health and performance of cattle is described elsewhere  was selected for DNA isolation and characterization by 16S rRNA gene sequencing . The available number of samples from chronically ill and mortalities did not total 50 for all vaccine treatment groups, and the remaining allotment for those samples was reassigned to the healthy and BRD case categories  using a Qubit 4 fluorometer (Thermo Fisher Scientific). After extraction, DNA was stored at −80°C until amplification was performed.A commercially available extraction kit  was used to isolate genomic DNA from DNS samples according to manufacturer instructions. Following isolation, DNA was quantified , low response (0 < log2BRSV titer≥4), and high response (5 ≤ log2BRSV titer≥ 9).From these values, animals were categorized into three groups for community-wide analysis: no responseThe M:M ratio was calculated with the following equation:Mycoplasma and Moraxella was from the same community .The RA (%) of both 2(BRSV titer) from day 0 to day 28, a linear model was fitted using all combinations of the log(M:M ratio), treatment group (INJ or INT), and the changes in RA of all genera representing at least 1% of the overall community  across all samples and the best model was chosen based on corrected Akaike's information criterion (AIC). A Shapiro–Wilk test and quantile–quantile plots were used to test data for normality and Breusch–Pagan tests were used to test for homoscedasticity with an alpha of 0.05. Mean separation was evaluated using a critical alpha level of 0.05.Unless indicated otherwise, R version 4.1.0 . Among animals that remained healthy throughout the study, richness and diversity were not significantly different among the three vaccine treatment groups  on d 0 or d 28 . However, richness increased between d 0 and d 28 within the CON group (P = 0.01), and diversity increased between d 0 and d 28 in the INJ and CON groups . An increase in richness or diversity was not observed (P = 0.36) for INT between these dates.There were no significant differences in the richness or diversity of respiratory microbiome when comparing animals administered either to the vaccine treatment group (INJ or INT) or to the control group on d 0 or d 28 . Ordination with NMDS also illustrated that bacterial communities did not differ among study groups .The microbiome composition did not differ significantly on d 0 or d 28 among the three vaccine treatment groups when analyzing data from all animals enrolled in the study or within animals that remained healthy  and INJ (p = 0.05) treatment groups (n = 35–43).To account for the impact of morbidity on the URT microbiome in differently vaccinated cattle, the rank of phylum was used to visualize the taxonomic composition of the microbial communities from healthy animals on d 28. In that population, Firmicutes  was the predominant phylum in animals from the CON and INJ group, but Proteobacteria  was most abundant within INT . InteresMoraxella was the most predominant genus followed by Mycoplasma, and combined the two genera comprised over 50% of the microbial community in all vaccine treatment groups . Across all animals on d 28, Mycoplasma was the most abundant genus followed by Moraxella . However, Histophilus was in higher RA within INT animals compared to CON and INJ at d 28, while Mannheimia was of significantly lower RA for INT compared to CON . To account for potential impacts associated with morbidity, the RAs of genera comprising at least 1% of the overall community were visualized and compared among healthy animals at d 28. In healthy animals, Moraxella was the most abundant genus within the INT group, while Mycoplasma was the most abundant in both CON and INJ animals , and Mycoplasma was significantly less abundant in healthy INT animals compared to healthy CON and INJ on d 28 . Furthermore, Mannheimia was in significantly lower RA in healthy INT animals compared to healthy CON animals, and Pasteurella was in significantly lower RA in healthy INT animals compared to both healthy CON and healthy INJ animals on d 28 . Given the overwhelming predominance of Mycoplasma and Moraxella, and the importance of Mycoplasma in BRD, a ratio of Mycoplasma:Moraxella RAs (M:M ratio) was calculated and compared between vaccine treatment groups in healthy animals at d 0 and 28. Animals in the INT group had significantly lower M:M than the CON group at d 28 (P = 0.03), but the M:M ratio increased in all vaccine treatment groups over time .To examine taxonomic differences at a more granular level among study groups, RAs for genera comprising at least 1% of the overall community were visualized and compared on d 0 and d 28. At d 0, t groups . There woraxella . Althoug animals . Moraxeln = 9–67, P = 0.46). However, based on generalized UniFrac distances, a significant difference in day 28 community structure was detected between communities from animals that had no change in log2 BRSV titer vs. those classified as high responders . The change in BRSV titer from d 0 to d 28 was best explained by changes in the RA of Mycoplasma . Differences were notable in average RA for Mycoplasma and Moraxella between animals that had a high vs low log2 BRSV titre change, but the variability in responses was quite large and these differences were not statistically significant , the log M:M ratio, or vaccine treatment were significantly associated with differences in the BRSV antibody response categories.There were no significant differences in the RA of any of the nine dominant genera between animals that were seropositive vs. seronegative to BSRV on d 28 . Diversity also increased significantly over time in BRD-affected animals but not in healthy animals or mortalities . Similarly, the overall composition of microbial communities on d 0 did not differ among cattle in different health classification groups, but community composition between BRD cases and healthy animals differed significantly on d 28 . Visualization with NMDS illustrated that microbial communities in d 0 samples did not differ based on final health classification, but there was a subtle divergence in community structures between healthy animals and BRD cases on d 28 , but there were no significant differences associated with other predominant phyla. On d 28, BRD cases had higher RA of Firmicutes and lower RA of Proteobacteria than cattle that remained healthy . At the rank of family, the RA of Mycoplasmataceae increased from d 0 to d 28, particularly among BRD cases . Other important BRD pathogens including Mannheimia, Pasteurella, and Histophilus either represented < 1% of the overall community or did not differ on d 0  among healthy cattle, BRD cases, and animals that died. At d 28, BRD cases and individuals that died had higher RAs of Mycoplasma than healthy cattle . Animals that remained healthy also had higher RAs of Moraxella than BRD cases at d 28 . Higher RA of Mycoplasma and lower RA of Moraxella was also seen for BRD Cases with fevers (CIS = 3) compared to those that did not have fevers .The increased abundance of Firmicutes was largely the result of an increased RA of the genus CIS = 2; . The RA n = 42–116, P = 0.03). At d 28, healthy animals continued to have a lower M:M ratio than BRD cases, but small sample size and large variation in M:M ratios within the remaining cattle that would go on to die resulted in a lack of statistical difference for that group .Interestingly, at d 0, animals that remained healthy throughout the study had a lower M:M ratio than animals that became a BRD case or died  at the genus level that exceeded abundance at BRD treatment than at d 0 or d 28 . At the time of initial BRD treatment, the RAs of Proteobacteria  and Moraxella  were both significantly lower than at d 0 but did not differ from d 28 .Samples collected at the time of the first BRD treatment had the greatest RA of Firmicutes  at the phylum level and n = 8–116, healthy P = 0.001, BRD cases P = 0.001, mortalities P = 0.002).To examine potential temporal shifts in the microbiome over the first 28 d in the feedlot, the composition of URT microbial communities was compared between d 0 and d 28 for animals within each health category . Ordination with NMDS illustrated that communities clustered by timepoint  for animals in all three health categories, suggesting there was a shift in URT community composition over this time, independent of vaccination or health status, and PERMANOVA confirmed this shift was significant . To investigate these temporal shifts further, differences in the Ras of genera comprising at least 1% of microbial communities were compared between samples collected at d 0 vs. d 28 for each health status. There were significant shifts in the RAs of all but 2 of the 11 most abundant genera in cattle that remained healthy and those that developed BRD . While similar trends were observed among cattle that died, owing to small samples sizes and large variation at d 28, only the increase in Mycoplasma, and the decrease in Mannheimia and unclassified members of Moraxellaceae were statistically significant in cattle that died during the study .In addition to shifts in the overall composition, the RA of different phyla also changed between d 0 and d 28. The most predominant phylum, Firmicutes, doubled in mean RA from 25.71% ± 1.41 (mean RA ± SEM) at d 0 to 52.60% ± 1.95 at d 28, while the second most abundant phylum Proteobacteria significantly decreased in RA from 62.53% ± 1.55 on d 0 to 39.12 ± 1.93 on d 28 . These differences in M:M ratio were also evident at d 0 in cattle that developed BRD or died, suggesting that these taxa may play an important role in the development of BRD or may be important biomarkers that could be used in the prediction of health outcomes for BRD to better target interventions, such as the use of antimicrobial metaphylaxis. Regardless of vaccination or health status, the URT microbial community also demonstrated a clear temporal shift in composition between d 0 and d 28 at the feedlot.Using 16S rRNA gene sequencing, this study found that the URT microbiome differed in cattle that were vaccinated intra-nasally with MLV targeting respiratory viruses compared with cattle that were vaccinated parenterally against the same viruses or unvaccinated CON cattle. However, changes in serum antibody titers to BRSV were not associated with differences in URT microbiome composition. Together, these findings suggest that changes in URT microbial communities were mediated by local immunity or interactions among microbial features within the respiratory tract as opposed to being mediated by systemic immune responses. Perhaps it is most notable that cattle vaccinated intra-nasally had lower RAs of Mycoplasma, which was also observed in a different subset of these same cattle when analyzed using rtPCR  were excluded from subsequent analysis in Powledge et al. (Mannheimia and Pasteurella). A reduction in these well-known BRD pathogens suggests that INT vaccination may have impacted the respiratory microbiome beneficially. Yet, Powledge et al.  within the perspective of entire URT microbial communities.Intranasal vaccination has been reported to be associated with changes in the respiratory microbiome in humans, mice, and pigs  can be found below: The animal study was reviewed and approved by West Texas A&M University Animal Care and Use Committee.JR and PM conceived and oversaw the research and provided internal research funding. JR, TM, and SP oversaw cattle management, experimental treatments, and sample collection. PM and CW oversaw and directed all laboratory activities. TM and SP conducted laboratory procedures to purify DNA and prepare amplicon sequencing libraries. LP, TM, PM, and JR planned bioinformatics and statistical analyses and were responsible for the interpretation of results and manuscript planning. TM and LP conducted statistical analyses. TM prepared initial drafts of the manuscript. All authors reviewed, edited, and approved the final manuscript version.


Another important finding was the central role of information insufficiency as a mediator/driving force in subsequent systematic information processing and protective behavior. Conclusions: The study has made important contributions in that it extends the scholarship on health information behaviors by (a) highlighting that relevant hazard experience in risk information seeking and processing model should be expanded to include indirect experience, and (b) providing the mechanism of subsequent systematic information processing following prior information processing. Our study also provides practical implications on health/risk communication and protective behaviors’ promotion in the pandemic context.Background: Few existing studies have examined information processing as an independent variable to predict subsequent information behaviors in a pandemic context, and the mechanism of subsequent information behavior processing following the initial or prior information behavior is unclear. Objective: Our study aims to apply the risk information seeking and processing model to explain the mechanism of subsequent systematic information processing in the context of the COVID-19 pandemic. Methods: A three-wave longitudinal online national survey was administered during the period of July 2020 to September 2020. Path analysis was conducted to test the relationships between prior and subsequent systematic information processing and protective behaviors. Results: One important finding was the key role of prior systematic information processing, as indirect hazard experience was found to be a direct predictor of risk perception ( Information behavior refers to information users’ active or passive information seeking, processing, or management . TheoretHowever, in a pandemic context, initial information behavior, can also be viewed as an exogenous variable that may predict future information behavior since the information behavior is an ongoing dynamic process that has different stages . As demoThe outbreak of the coronavirus disease 2019 (COVID-19) in China, an infectious disease caused by a novel virus called SARS-CoV-2, provides an optimal context to examine the process of subsequent information behavior due to several reasons. First, people rely heavily on adequate information, especially online information, during emergency situations to make decisions and perform preventive behaviors . Second,Given that few existing studies have examined information processing as an independent variable to predict subsequent information behavior in a pandemic context, we proposed a longitudinal study to address the issue. A longitudinal study design was chosen over a cross-sectional design because it would enable us to conduct lagged analyses to establish temporal order. Applying the RISP model , complemTwo comprehensive frameworks to explain the mechanism of information behavior—the RISP model  and WilsHowever, the RISP model  did not As one of the central constructs of RISP , risk peInformation processing as a type of information behavior can be further categorized into systematic and heuristic processing , which aSystematic processing focuses on in-depth consideration of the content of messages and can be viewed as a process of vicarious learning  or indirThe RISP model  posits tH1. Direct experience will positively predict risk perception.H2. Prior systematic information processing behavior as indirect experience will positively predict risk perception.Trust has played an important role during the pandemic because it may influence people’s risk perception and their behaviors in response to the pandemic  ,25. GeneH3. Higher levels of trust in local government’s risk management predict lower risk perception.Negative affective responses  ,11 are oH4. Risk perception positively predicts negative affective responses.H5. Negative affective responses positively predict systematic processing.Information insufficiency is the knowledge needed, in addition to the existing knowledge one has , or “perH6. Negative affect positively predicts knowledge insufficiency.H7. Information insufficiency positively predicts systematic information processing.H8. Prior systematic processing as indirect experience positively predicts information insufficiency.According to RISP, informational subjective norms represent “perceived social normative influences motivating the desire for information sufficiency” . If peopH9. Systematic processing subjective norms positively predict information insufficiency.H10. Systematic processing subjective norms positively predict systematic information processing.H11. Systematic information processing positively predicts protective behaviors.The model including the hypotheses is presented in RQ: What is the indirect effect between (a) direct experience, (b) indirect experience, and (c) trust in risk management and protective behaviors?This study was approved by the Institutional Review Board at a university in China. Participants were adults (18 and older) who resided in mainland China since the start of the pandemic. A three-wave longitudinal online national survey was administered among potential participants by a professional survey company  during the period of July 2020 to September 2020. The decision to start the survey on July 2020 was based on two facts: 1. By the end of June, COVID-19 had rapidly spread to other countries and become a global pandemic . 2. On 2We collected 871 responses at T1, 613 at T2, and 437 at T3, respectively. The participants with three waves of 100% completed surveys and two correct attention check  items weFor the longitudinal survey, participants’ sociodemographic variables  were measured at T1. RISP constructs and protective behaviors were measured at T1, and/or T2, and/or T3. The original questionnaire was in English and was translated to Chinese. The Chinese version was then back translated into English and was pretested before survey administration to ensure the clarity and readability of the questionnaire. For the pretest, we invited 10 Chinese native speakers to fill out the Chinese version of the questionnaire. Then, we asked them about the clarity and readability of the questionnaire and made changes accordingly. We then invited another five Chinese native speakers to fill out the revised questionnaire, and no one reported any clarity or readability issues.Direct experience was measured at T1 through two items adapted from existing studies ,44,45. SProtective behaviors , adapted from an existing study  as well To test the direct effects, path analysis was used to test the constructs (average scores were computed for each construct) of RISP and protective behaviors, with systematic processing and protective behaviors at T1 or T2 controlled in the analyses. Statistical software Mplus 8.0  was emplThe mean age of the participants was 33.81 , and 46% participants were female. The majority (67.90%) of the participants had a bachelor’s degree. Please see 2 = 71.44, df = 49, p = 0.02, RMSEA = 0.035, CFI = 0.96, TLI = 0.95, SRMR = 0.06) indicated good fit to the data; even the p value was significant. Direct experience , indirect experience , but not trust in risk management , significantly predicted risk perception, which supported H1 and H2 but not H3. That risk perception significantly predicted affective responses  at T1 supported H4. Affect , indirect experience , but not systematic processing subjective norms , significantly predicted information insufficiency at T1, which supported H6 and H8 but not H9. Information insufficiency , and subjective norms , but not affect  at T1, significantly predicted systematic information processing at T2 after controlling for T1 systematic information processing; therefore, H7 and H10 were supported, but H5 was not supported. Systematic information processing  at T2 significantly predicted protective behavior at T3 after controlling for T2 protective behaviors; thus, H11 was supported. Please see The results for the overall fit of proposed model (χTwo significant indirect paths from indirect experience to protective behaviors were identified through (a) systematic processing , and through (b) insufficiency and systematic processing as serial mediators . All other indirect effects were not significant , nor did it predict protective behaviors indirectly in our model. Our interpretation is that trust in risk management can be an important predictor of initial information behaviors, but our study focused on explaining subsequent information behaviors. Trust in risk management may not be a driving force of subsequent information behaviors. Alternatively, it might be a key predicting factor for subsequent information behaviors, but it lacked variability in our data and people’s trust in risk management during COVID-19 was all relatively high in China. Therefore, more studies are needed to test the role of trust in risk management. The fact that H5 (from affect to systematic processing) was not supported but H7 (from knowledge insufficiency to systematic processing) was supported indicated the aforementioned central role of information insufficiency. The fact that H9 (from norms to knowledge insufficiency) was not supported but H8 (from indirect experience to knowledge insufficiency) was supported indicated the aforementioned key role of prior systematic information processing.The findings of the current study advance the theoretical frameworks from three perspectives. First, relevant hazard experience, as a construct of RISP , should The findings also have practical implications. With the goal of protecting people from contracting COVID-19 and exercising protective behaviors, measures and strategies that emphasize the importance of ongoing systematic information processing and help motivate people to systematically process online information from credible sources should be promoted. For instance, for credible health information websites , a section on the importance and the potential benefits of ongoing systematic information processing can be provided. Given that mass media were indicated as the dominant information source for the pandemic , the impOur study has several limitations. First, the study was conducted using non-probability sampling, although we used a professional survey company to collect data nationally. Therefore, the sample might not be representative of the population. Future studies could replicate the current study using probability sampling. Second, the RISP model  has inclIn sum, guided by the RISP model  and Wils


P = 0.008). Neither lighted incubation nor larvae provisioning affected fearfulness, feather pecking, plumage condition or recovery time after vaccination. In conclusion, the present study showed no effects of light during incubation and minor effects of foraging enrichment during rearing on the behavior of laying hen pullets. Further research is recommended on other welfare aspects.Severe feather pecking, the pulling out of feathers of conspecifics, is a major welfare issue in laying hens. Possible underlying causes are fearfulness and lack of foraging opportunities. Because early life is a crucial stage in behavioral development, adapting the incubation and rearing environment to the birds' needs may reduce fearfulness and prevent the development of feather pecking. In a 2 × 2 factorial design study, we investigated whether a green light-dark cycle throughout incubation, which resembles natural incubation circumstances more than the standard dark incubation, and foraging enrichment with live larvae during rearing reduce fearfulness and feather pecking and increase foraging behavior of laying hen pullets from an early age onwards. In this 2-batch experiment, 1,100 ISA Brown eggs were incubated under either 0 h of light/24 h of darkness or 12 h of green LED light/12 h of darkness. After hatching, 400 female chicks (200 per batch) were housed in 44 pens (8–10 chicks per pen). During the entire rearing phase (0–17 wk of age), half of the pens received black soldier fly larvae in a food puzzle as foraging enrichment. We assessed fear of novel objects and humans, feather pecking, plumage condition, foraging behavior, and recovery time after a 3-fold vaccination (acute stressor). A slight increase in the number of foraging bouts was only seen with larvae provisioning (rate ratio 1.19, 95% CI 1.02–1.29,  FP). FP is defined as the pecking at and pulling out of feathers of conspecifics, leading to pain, baldness, injuries, and even mortality. This behavior is, therefore, a major welfare issue in the poultry industry. FP has multiple underlying causes, including fearfulness and lack of foraging opportunities  has been to routinely beak-trim laying hen chicks, that is, remove the tip of the beak to prevent damage caused by FP. Due to societal and ethical concerns, however, this procedure is now banned in the Netherlands and several other EU countries. It has been prohibited in organic husbandry since 2008 (EC No 889/2008). Because of the ban, the risk of FP outbreaks is again a problem and keeping hens with intact beaks can result in more severe damage than keeping flocks with trimmed beaks , could potentially be used as effective environmental enrichment for laying hen pullets because insects are part of the natural diet of chickens and their movement is highly attractive to these birds. BSFL have a unique nutritional profile and they can be used efficiently in the sustainable breakdown of organic waste material .This experiment was approved by the Dutch Central Authority for Scientific Procedures on Animals (CCD) under license number AVD1080020198685, and by the Animal Welfare Body Utrecht under work protocol numbers 8685-1-01 and 8685-1-03. The study was executed in accordance with the Dutch legislation and the EU directive on animal experimentation.DnL]; dark, larvae [DL]; light, no larvae [LnL]; light, larvae [LL]. Chicks were randomized and housed in 44 pens . Treatments were not mixed within pens. Due to a randomization error, the treatments were unequally divided over the pens in batch 1 . To compensate for this unequal sample size, 4 extra pens were built to increase statistical power in batch 2 (7:5:5:7). Since there were 200 laying hen pullets per batch, group sizes per pen differed between the batches . For both batches combined, the total numbers of pens per treatment were 10:12:12:10. To avoid incubator effects, approximately equal numbers of chicks from incubators with the same light treatment were housed within a pen.This experiment was conducted in 2 batches and used a 2 × 2 factorial arrangement combining light vs dark incubation and presence vs absence of larvae during rearing, 4 treatments were defined: dark, no larvae [A total of 1,100 eggs of the layer hybrid ISA Brown  were incubated at the research facility of Wageningen University & Research in Wageningen, NL, in 2 batches . Logistically it was not possible to perform both batches in the same season, because the research facility was unavailable in that period. Age of the parent stock was 43 wk and 34 wk, respectively. In each batch, eggs were randomly assigned to 1 of 2 conditions: Light or Dark incubation. The Light eggs were exposed to 12 h of green LED light and 12 h of darkness per day, whereas the Dark eggs were not exposed to any light during incubation. There were 2 incubators per treatment; 1 HatchTech incubator with an egg set capacity of 1,400 eggs, and 1 climate respiration chamber with an egg set capacity of 400 eggs. For more information about the incubators, see The rearing pens were each 246×88×241 (l×w×h) cm, separated by wire mesh and a 60-cm high wooden barrier to prevent visual contact between adjacent pens. The pen floors were initially covered with peat as litter material to create a larger visual contrast with the larvae. The peat was replaced with wood shavings from wk 8 onwards after the occurrence of severe eye infections in 2 chicks . The number of larvae provided corresponded with 10% of the daily feed intake as described in the ISA Brown product guide, and therefore increased with age. Tailor-made diets  gradually replaced the commercially available diet from 7 d of age onwards. This diet included additional protein and BSF oil in the diet of the no-larvae pullets , to compensate for any nutritional effects caused by the 10% larvae feeding in the DL and LL groups. The larvae were provided in transparent cylinders (15 × 4 cm) containing three 9-mm holes each. This design was based on a previous study on broilers . A pilotAll behavior tests described in the present study were performed at pen level. Plumage condition was scored per individual pullet. Observers were blind to the incubation treatment, but not to the larvae treatment, as larvae dispensers were present during the observations. Detailed test protocols, as well as the corresponding raw datafiles, are available in the DANS repository (DOI: 10.17026/dans-26r-bywc). A timeline of all measurements described in the present study is provided in Novel Object Test. The response to a novel object (NO) was assessed at 6 d (NO1) and 10 wk (NO2) of age. After sitting in front of the pen for 5 min as habituation, the observer placed a NO in the middle of the pen floor. The latency for the first pullet to approach the object within a 3-chicken distance was scored. The test duration was 4 min. During NO1, the object was a rectangular-shaped piece of wood, covered with tape in multiple colors. During NO2, the object was a beverage can. The observation protocol was adapted from Human Approach Test. Fear of humans was measured at 10 wk of age during a human approach test (HA). The observer stood stationary at the pen entrance for 5 min, and the latency of the first 3 pullets to approach was recorded (N = 44).Behavioral Recovery after a Stressor. Behavioral recovery data were collected for the pullets in batch 2 only. At 13 wk of age, all pullets received 3 consecutive vaccinations, which were a wing web vaccination (fowl pox), an eye drop vaccination (infectious laryngotracheitis), and an intramuscular vaccination in the pectoral muscle . To assess whether or not the treatments affected the recovery time after an acute stressor, chicken behavior was scored at pen level for 60 min after vaccination, using scan sampling with 10-min intervals  and severe (SFP) feather pecks were defined according to this age . Pecks wPlumage Condition. At 15 wk of age, plumage condition was scored according to Foraging Behavior. Foraging behavior was observed at 1, 4, and 7 wk of age  in case of count data and hazard ratios (HR) in case of latency data, both with 95% CI. A 95% CI covering value 1 (unity) means there was no difference between the means of the 2 conditions. Effects are considered significant at P ≤ 0.05. Plumage condition was analyzed with Bayesian multinomial logistic regression using Stan . Light during incubation did not affect latency to approach the NO at 6 d of age . Similarly, the NO test at 10 wk of age showed no effects of light during incubation  or larvae provisioning . The latency to approach a familiar human in the HA test was not affected by either light during incubation  or larvae provisioning .Behavioral Recovery after a Stressor. Light during incubation and larvae provisioning did not affect behavior in the 60 min after the 3-fold vaccination compared with behavior on the baseline days (P > 0.05). Irrespective of treatment, preening behavior increased immediately after vaccination  nor larvae provisioning during rearing  affected the number of GFPs  or larvae provisioning . In addition, no effects were found of lighted incubation  or larvae provisioning  on total foraging duration.Batch Effects. Batch effects were found in the NO test, FP, and Plumage Condition scores. Though batch effects are outside the scope of this study, these results are available in the supplementary data as a contribution to the transparency and reproducibility of animal experiments.This study investigated the effects of a green LED light-dark cycle throughout incubation and BSFL provisioning during rearing as interventions to reduce fearfulness and the development of FP throughout the rearing phase of laying hens. Neither lighted incubation nor larvae provisioning seemed to affect these behaviors. Larvae enrichment did slightly increase foraging frequency, but not foraging duration.No effects of lighted incubation or larvae provisioning were found on the latency to approach a NO or a human. The overall latencies to approach were similar to those found in previous studies . In commLighted incubation and larvae provisioning did not affect FP or plumage score. The pullets in our study rarely showed SFP. Although this damaging behavior usually develops at the onset of egg-laying , some stLarvae-enriched pullets initiated more foraging bouts than nonenriched pullets, but total foraging duration was not affected by larvae provisioning. These results imply that foraging enrichment causes more frequent, but shorter moments of foraging. Despite the known association between the provision of sufficient foraging opportunities and reduced FP, the increased number of foraging bouts was not related to lower levels of FP in this study.This experiment is the first to report on the effects of provisioning BSFL as enrichment throughout the rearing phase of laying hens. Existing literature has been clear about the positive effects of enrichment on the reduction of fearfulness and FP, and the promotion of positive behaviors such as foraging . Given the limited beneficial effects of larvae as enrichment in the present study, ethical and environmental considerations need to be addressed before applying larvae enrichment on a commercial scale. First, larvae should be reared in a way that does not negatively impact their own welfare . Second,This study did not show any significant effect of exposure to a green LED light-dark cycle throughout incubation or larvae provisioning on fearfulness, early FP, or plumage score in laying hen pullets. The only significant treatment effect was an increased number of foraging bouts when pullets were provided with larvae. Despite the lack of effects in the current study and given the body of literature that suggests a positive impact of both lighted incubation and enrichment during rearing on behavioral and physiological traits, additional research is recommended to further elucidate the full potential of these strategies to improve laying hen welfare.the European Union's Horizon 2020 Research and Innovation Programme under grant agreement N°816172. The authors would like to thank Inge van den Burg, Casper van Eekelen, Dylan Geerman, Margaux Laurent, Elyse van Leeuwen, Lotte Pluim, Antoine Prunier, Arjen van Putten, Lisa Veldkamp, and Jary Weerheijm for their help with the data collection; Jan van den Broek for additional statistical support; Marcel Heetkamp for the incubation process; Freek Weites and Marc Kranenburg for taking care of the pullets; and Mieke Matthijs and Thijs Manders for providing veterinary care.This project received funding from T. B. Rodenburg reports financial support was provided by Horizon 2020 Research and Innovation Programme. The remaining authors declare no conflicts of interest.


Chemical exchange kinetics of microplasticswere measured for 10 polychlorinated biphenyls (PCBs) as proxies forthe broad class of hydrophobic organic chemicals. Following earlierstudies, olive oil was used as a proxy for digestible food, underhigh and low digestive enzyme activities. The micelle–waterand oil–water partition coefficients of the 10 PCBs were alsodetermined to evaluate the relative contribution of each gut componentto sorb PCBs. A new biphasic and reversible chemical exchange model,which included the digestion process, fitted well to the empiricaldata. We demonstrate that the digestive processes that break downcontaminated food can lead to a substantial increase in chemical concentrationin microplastics by a factor of 10–20, thereby reducing theoverall chemical bioavailability in the gastrointestinal tract whencompared to a scenario without microplastics. Higher enzyme activitiesresult in more chemicals being released by the digested food, therebyresulting in higher chemical concentrations in the microplastics.While the model-calibrated kinetic parameters are specific to thestudied scenario, we argue that the mechanism of the reduced bioavailabilityof chemicals and the modeling tool developed have generic relevance.These digestive processes should be considered when assessing therisks of microplastics to humans and also biomagnification in aquaticfood webs.The transfer kinetics of plastic-associated chemicalsduring intestinaldigestive processes is unknown. Here, we assessed whether digestiveprocesses affect chemical exchange kinetics on microplastics, usingan  New biphasic and reversiblechemical exchange model withdigestion processes to understand the human health effects of microplastics. They may alsobe inhaled from air due to their prevalence in the environment. Humaninteraction with MPs is inevitable, as we depend on plastic materialsin almost every part of our lives . Over an average human lifetime of 70 years, it is estimatedthat an adult can be exposed to up to 20 million particles (1–5000μm), which is equivalent to 12 mg.9 These estimations were based on nine intake media that were knownto have MPs and comprise 20% of the human diet. MPs ingested by humanseither come from the abrasion and wear-and-tear of plastic productsused in our daily lives to process, transport, and store food andbeverages or come from bioaccumulation in the food chain .10All biota, includinghumans, are exposed to MPs (<5 mm).11 An established approach to investigate the chemical vector effectof MPs when ingested is by performing in vitro gutfluid experiments. This type of experiment has been widely used toinvestigate chemical bioavailability from indigestible particles suchas sediment, asphalt, and soot in human and animal digestive tracts.16 Such studies have been increasingly favored to understand the MPvector effect in seabirds, aquatic organisms, and invertebrates fora variety of chemicals.21 Bioaccessibilities of both organic compounds and heavy metals havealso been investigated in different types of in vitro setups mimicking the human digestive tract.24 However, dueto the nature of the experiments, these studies can only elucidatethe key physicochemical properties of the physiological fluids andpolymers that may have affected the chemical bioaccessibility fromMPs in each compartment of the digestive tract but not the role ofother compartments present, such as food.As a carrier for a mixture of chemicals,these plastic particlesmay release chemicals in the organisms’ digestive tracts wheningested, potentially increasing exposure and risks.in vitro studies are alsouseful for estimating chemical exchange kinetics.25 Using chemical kinetic rate constantsand other sorption parameters, the chemical behavior has been simulatedfor different gut retention times to estimate the chemical bioaccessibilitiesfrom ingested plastic in different species.21 Yet, although the aforementioned study had included food when investigatingthe chemical exchange in MPs, the food component was an inert pool.21 In reality, this is not the case since foodis digested, and it is uncertain if the digestion dynamics will affectthe chemical exchange kinetics in MPs. It has been well-establishedthat food digestion and absorption in the gastrointestinal tract (GIT)are important factors in chemical uptake from food.27 However, we are uncertain whether this is the case in the presenceof MPs.Besides measuringchemical bioaccessibility, 27 Hence, this increases the chemical fugacity of the GIT content abovethat of the consumed food, which has been demonstrated to be the drivingforce behind gastrointestinal magnification.27 We speculate that in the presence of MPs, the chemicals associatedwith food in the GIT will transfer from the higher fugacity in thefood to the lower fugacity in the MPs and other gut constituents.31 Therefore, MPs may act as a sink and potentially attenuate chemicalbiomagnification in the biota when the MPs pass through the GIT andare excreted out.21 To evaluate if MPs act as a majorsink for the chemicals and significantly reduce chemical bioaccumulation,the chemical behavior needs to be assessed across all gut componentsas the food is digested. This includes quantifying the partition coefficientsof the chemicals between the other gut components such as food andmicelles (which are either produced in the gut or formed from thebreakdown of food) and the aqueous phase.When food is digested in the gastrointestinal tract,the compositionof the food changes, reducing the chemical fugacity capacities ofthe GIT content to be below that of the consumed food.32 and low-density polyethylene (LDPE) strips representingingested MP particles were pre-equilibrated with a range of PCBs toachieve realistic gut chemical conditions. LDPE was chosen here asit is one of the highly produced and used polymer types33 and is thus a good proxy for microplastics ingeneral. PCBs were used as model hydrophobic organic compounds (HOCs)because they are commonly found in environmental plastics36 and in food38 and can represent the generalchemical behavior of HOCs. The chemical dynamics of PCBs in the LDPEwere then examined upon lipid digestion as initiated by the additionof lipase under high and low enzyme kinetics, respectively. Polyoxymethylene(POM) passive samplers were used to determine the aquatic phase measurementsof PCBs without separating the nonplastic gut fluid components.41 A previously well-accepted dynamic multicompartment model was redesignedto include the digestion process.21 Thechemical kinetic parameters for HOCs on MPs during food digestionare discussed in the context of chemical bioavailability and rigorousrisk assessments of plastic-associated chemicals for humans and otherphysiologically similar biota.9Therefore, in thepresent study, an environmentally realistic scenariowas mimicked, whereby an organism ingests contaminated MPs and contaminatedfood, followed by food digestion. The aim was to quantify the kineticrates of chemical transfer to and from microplastics and elucidatethe influence of intestinal digestive processes, in this case, lipiddigestion as a proxy for food digestion, on the overall chemical bioavailabilityunder simulated gut fluid conditions. To this end, batch setups ofsimulated gut fluids mimicking the upper human gastrointestinal (GI)tract conditions containing olive oil as food lipidsKOW)42 ranging from 5.24 to 8.18 were prepared in individualstock solutions in either acetone or isooctane at ∼20–100mg/L. The concentrations of each PCB congener in the spike mixtureand the final experimental setup are provided in the Supporting Information (SI) in Table S1. LDPE strips witha thickness of 30 μm were cut from zipper bags found in thesupermarket, which were also used for our previous experiment.21 They were cut to a final size of 5.1 ×5.1 cm. As the thickness of the strips was 30 μm, this representsthe intrapolymer diffusion path lengths similar to those of 30 μmspherical or irregularly shaped environmentally relevant microplastics.21 POM strips of thickness 76 μm were obtainedfrom CS Hyde Co.  and cut into strips with a sizeof 3.2 × 3.2 cm to be used as equilibrium passive samplers toassess the aqueous phase PCB concentrations after 28 days.43 Both LDPE and POM strips were cleaned in methanol (MeOH) for 24h under constant shaking on a horizontal shaker at 150 rpm and thencleaned with Milli-Q water for another 24 h to remove any residualMeOH and air-died before use. Olive oil was purchased from the supermarket. Reagents used for preparing thesimulated digestive fluids include sodium taurocholate (NaTC) , bovine serum albumin (BSA) , sodium chloride (Merck Millipore), and sodium azide. Lipase FE-01 (>18 000 U/mL) was obtainedfrom ASA Specialenzyme GmbH, Germany.All chemicals used were of analytical gradeor higher purity, and solvents were high-performance liquid chromatography(HPLC) grade. Twelve PCB congeners  with log octanol–waterpartition coefficients , which initiated lipid digestion. Three treatments were set upto simulate high  and low (heat-inactivatedlipase) enzyme activities and a control treatment to account for anybackground PCBs from gut fluid components (heat-inactivated lipasewith no PCB spike). The high and low enzyme treatments are to comparebetween high and low metabolic digestive systems, respectively, sinceall humans have different metabolic rates.45 Olive oil (7 g), 10 LDPE strips , and a POM passive sampler (0.11 g) were added to each bottlefilled with ∼69.9 mL of gut fluid. Note that the mass of plasticused in this experiment may or may not represent the mass of plasticthat humans eat daily. This does not disqualify the results, as thetargeted kinetic parameters are independent of the mass of plastic.Furthermore, a certain minimum mass of plastic is necessary to meetthe chemical analytical minimum detection limits. Following previousstudies, to limit biodegradation of the olive oil, sodium azide wasadded to a final concentration of 25 mg/L.46 This is 0.002 wt % of the aqueous phase, which is lower than thecommon range (0.02–0.04%) for inhibiting microbial growth inenzyme assays and thus should not affect the lipase digestion process.48 The final pH of the mixture was 6.08. Each bottle from the highand low enzyme treatments was spiked with 100 μL of PCB stockmixture (Table S1) and shaken on a horizontalshaker at 150 rpm at 37 °C for 28 days to allow the chemicalsto partition to the different components in the gut fluid mixture.This is to mimic an environmentally relevant scenario of chemicalintake by humans via both food and MP ingestion. It also mimics analready contaminated human gut due to chemical bioaccumulation inthe body over the years. The final concentrations of individual PCBcongeners in each system ranged from 30 to 124 μg/L (mass ofPCB per volume of assay) (Table S1) toensure that equilibrium concentrations in plastic and food after 28days were environmentally realistic.49All experiments wereperformed in 100 mL transparent glass bottles with glass stoppersin quadruplicates. The simulated gut fluid mixture was prepared toachieve final  concentrationsof 10 mM of NaTC and 5 g/L of BSA, constituted in 150 mM sodium chloridesolution.41 PCB concentrations in POM are used to determine the PCB concentrationsin the aqueous phase. In addition, 200 μL of the gut fluid mixturewas sampled for lipid analysis. For the high enzyme treatment, 10mL of lipase was added to the bottles in its original form based onthe enzyme activity concentration from Minekus et al., 2014.50 On the other hand, for the low enzyme treatment,10 mL of lipase was heat-inactivated in a 14 mL glass centrifuge tubeby heating it to 100 °C in a water bath for 10 min. The heat-inactivatedenzyme was then added to the bottles. This was similarly done forthe control setups. The final food (olive oil and LDPE) to gut fluidratio was 1:10 (v/v)50 in the systems, with a 1:10 (v/v)ratio for plastic to olive oil. This ratio was chosen to representdigestive systems with a higher volume of lipid in comparison to ingestedplastic. The contents of the bottles were mixed by swirling with handsfor 5 min before removing one LDPE strip. Subsequently, the gut fluidkinetics experiment was carried out over 72 h with continuous shakingon a horizontal platform shaker incubator at 150 rpm and 37 °C.The experiment was performed for a longer time scale than realisticdigestion times. Over longer time scales, higher chemical concentrationgradients can be observed; hence, chemical transfer kinetic ratescan be assessed more accurately. Furthermore, this way, we also considerpossible scenarios whereby MPs are retained in the gut longer thanfood. Although there is a lack of empirical evidence on the gut retentiontimes for MPs in humans, other studies have shown that the MP retentiontime in animals’ guts, such as fish and lobsters, differs fromfood.53 One LDPE strip was removed after 2, 4, 6, 8, 10,24, 48, and 72 h of exposure to lipase and then cleaned with a lint-freetissue before storing in acetone-cleaned aluminum foils at −20°C until chemical extraction. Two hundred microliters of liquidemulsion were collected from the systems after 2, 4, 6, 10, and 72h of exposure to lipase and immediately transferred into a glass centrifugetube filled with 2 mL of ternary solvent prepared with dichloromethane,methanol, and MQ-water in a ratio of 1:2:0.8 (v/v/v) for solvent lipidextraction. Chemical analysis of the plastic strips was carried outbased on a previously published method.21 The methods for lipid extraction and analysis are provided in the SI. The rates of hydrolysis of lipids in thehigh and low enzyme treatments were calculated using molar concentrationsof free fatty acids (FFAs) produced in the test medium over time.The molar concentrations (μM) of FFAs liberated by lipase canbe determined directly from the measurements of fatty acids over timeor from the change in pH of the systems.55 The hydrolysis rates indirectly reflect the difference in the enzymeactivities between the high and low enzyme treatments.After 28 days, the POM passive sampler and one LDPE strip werecollected from each bottle by using metal forceps. Since partitioncoefficients for POM passive samplers are accurately known,56 and 1.7 mg/L,57 respectively.Hence, the concentrations used in this experiment surpassed the CMC,ensuring that micelles were formed. For the olive oil experiment,three levels of olive oil concentrations, 1.3, 6.3, and 12.6 g/L in8 mL of the gut fluid mixture, were prepared in glass centrifuge tubesalso containing one POM strip each. Each system was spiked with 10μL of the PCB stock mix and shaken at 250 rpm at 37 °Cfor 28 days, and all experiments were run in quadruplicate. After28 days, the POM strip was removed from each tube, cleaned with alint-free tissue, and analyzed for PCBs. The PCB concentrations onthe POM strip were then used to calculate the aqueous phase concentrationsin the system to estimate the partition coefficients of PCBs for micellesand oil using chemical partitioning theories.58 The partition coefficientsof PCBs between micelle and water  and between oil and water  can be obtained from the following equationsSI). Cinit is the total initial concentration of each PCB congener in the system(μg/L), KPOM is the POM-water partitioncoefficient (L/kg) obtained from Hawthorne et al. (2009),41CPOM is the PCBconcentrations in the POM strip normalized to the system volume (μg/L),[POM] is the concentration of POM in the system, i.e., mass of POMper unit of system volume (kg/L), and [micelle] and [oil] are themass of micelle and oil normalized to the system volume, respectively(kg/L). For a more detailed explanation, the reader is referred tothe SI (Determination of partition coefficients Kmicelle and Koil).In addition to the main experimental design, twoseparate experiments were designed to determine the micelle–waterand oil–water partition coefficients of 10 PCBs under varyingamounts of micelles and olive oil. Liberated free fatty acids fromolive oil can form micelles due to their amphiphilic structure. Therefore,for the micelle experiment, mixed micellar solutions were made withNaTC and increasing additions of oleic acid  to obtain total micelle-forming surfactant concentrations of5.7, 6.9, 11.6, and 64.5 g/L in 8 mL of the gut fluid mixture in glasscentrifuge tubes containing one POM strip each. The critical micelleconcentrations (CMCs) of NaTC and oleic acid are 8–12 mM21 The model was adapted to include the digestionprocess of food, and in this case, olive oil was used as a proxy.A schematic overview of the model is provided in SI Figure S1. The PCBs added to our gut fluid mimic systemare distributed into the different gut components. Based on chemicalmass conservation, the mass of chemicals in μg residing in water(MW), micelles (Mmicelle), oil (Moil), and the twoplastic types in the system, POM (MPOM) and LDPE (MLDPE), always remains equalto the initial total mass of chemicals We extendedan existing first-order multicompartment model to simulate the exchangeof chemicals between plastics, water, micelles, and food.Vw; L), this yieldsIf we divide all of the terms in Ctotal (μg/L)is the initial total concentration of chemicals, Cw (μg/L) is the chemical concentration in the water, Cmicelle ,and Coil (μg/L) are the concentrationsof chemicals in the micelle and oil phase, respectively, and CPOM (μg/L) and CLDPE (μg/L) are concentrations of chemicals in the POMand LDPE strips, respectively, all in terms of the volume of the system(VW). Note that all of the terms in Cmicelle*, Coil*, CPOM*, and CLDPE* are the chemical concentrations in the solid phases (μg/kg),and [micelle], [oil], [POM], and [LDPE] are the concentrations ofthe solid phases in the system (kg/L). Note that here, the superscript‘*’ indicates that concentrations are based on solid-phasemass to distinguish them from the concentrations in terms of systemvolume. At equilibrium, the solid-phase concentrations Cmicelle*, Coil*, CPOM*, and CLDPE* can be related to the aqueous-phase concentrationand the chemical partition coefficients Kmicelle, Koil, KPOM, and KLDPE  betweeneach of the phases and water, which leads toEach of these terms thus can be seenas a concentration expressedin terms of the same system volume, which illustrates the conservationof mass in the systemNote that all of the terms in 41 Hence, the chemical concentration in water at the startof the digestion process (t = 0 h), Cw, can be determined from CPOM* after the preincubationof 28 days and KPOM (L/kg)59It has been demonstrated that 76 μmPOM samplers approachequilibrium within 14 days at room temperature.KPOM were derived from apreviously established log–linear relationship with KOW.41Values of 60 and oil61 as comparedto the condensed crystalline POM matrix, and due to the higher temperature,we can safely assume that the chemical concentrations in micelle andoil also reached equilibrium after the preincubation period of 28days. Therefore, the chemical concentrations in the micelle and oilat the start of the digestion process (t = 0 h) canbe calculated with Kmicelle and Koil established from the experiments definingpartitioning coefficients of the micelle and oil, respectively  is released.55 The FFA molar concentrationsdisplay an exponential behavior over time with a limit at infinity(63 (kFFA isthe first-order rate constant (h–1) and [FFAmax] (μM) is the maximum molar concentration of FFA thatcan be liberated. FFA molecules tend to form micelles due to theiramphiphilic structure,64 and this thusincreases the micellar compartment of the system. The total mixedmicelles, [micelle]t (kg/L), because ofthe aggregated FFA and NaTC over time areFFA (g/mol) is the averagemolecular weight of the FFA based on the fatty acid composition measured(Table S2). On the other hand, while FFAmolecules are being liberated, the mass concentration of oil (triglycerides),[oil]t (kg/L), decreases over time. Thiswas calculated asoil (g/mol) is the molecularweight of olive oil triglycerides, which was calculated from the MWFFA (see eq S13 in the SI). Themolar concentration of oil triglycerides is equivalent to one-thirdof the molar concentration of FFA since a triglyceride (lipid) ismade up of three fatty acids.In the second phase of the experimentalsetup wherein lipase wasadded into the systems, a previously described biphasic kinetic modelsimulating the exchange of chemicals between plastics, water, micelles,and food componentsinfinity1. Therefnity. Therefore, the LDPE-water chemical exchangeis the overall rate-determining process.21 The PCB concentration in LDPE (CLDPE*) was modeled by fitting 21 (SI eqs S9 and S10) with the additionalaforementioned digestion kinetics, i.e., k3 (h–1), had a half-life of 13–32 days based on the previous study.21 Therefore, only the kinetic rate constants, k1 and k2, were fittedusing the minpack.lm65 package and deSolve66 in R. The fast desorption reservoir fractionof the total mass of chemical bound to plastic, f1 (dimensionless), and the intrapolymer rate constant, k3 (h–1), were set accordingto obtained relationships with KOW fromour previous study since the same LDPE strips were used.21 Boundary conditions for the PCB concentrationsin LDPE were based on the concentrations measured at t = 0 h whereby the slow and fast reservoirs of the polymer were assumedto be in equilibrium. The removal of LDPE strips at each time pointwas also accounted for in the modeling. However, the extraction ofthe emulsion was not accounted for since the total amount extractedonly comprised ∼1% of the total volume of the system.Following our previous study, for all systems,the boundary conditionfor Kmicelle and log Koil, respectively, analysis of covariance (ANCOVA) was usedto measure the main effect and interaction effects of the mass concentrationlevels of micelles and oil while controlling the effects of log KOW. The analysis was performed using ANOVA withType III sums of squares from the car67 package in R.68 Prior to the analysis,key assumptions were checked. First, independence between the covariate(log KOW) and treatment groups(mass concentration levels of micelles and oil) was tested with anANOVA model. Second, the variances among treatment groups were checkedfor homogeneity with Levene’s test found in the car67 package. Since the mass concentration levelsof micelles and oil had a significant effect while controlling thelog KOW variable, a post hoc multiplecomparisons was performed using the Tukey test (multcomp package69) to determine which treatment groups are differentfrom each other. ANCOVA was similarly performed to investigate theinfluence of the enzyme treatments on log k1 and log k2 whilecontrolling the effects of log KOW. ANCOVA was also used to investigate the differences between thelinear regressions (log Kmicelle vs log KOW) of Schacht et al.(2016)70 and our study.To investigate if the differentmass concentration levels of micelles and oil influenced the log 72 the effect of increasingconcentration of micelle-forming surfactants (NaTC and FFA) on themicelle–water distribution coefficients of the combined mixtureof micelles was investigated. The lowest micelle concentration (5.7g/L) was the baseline of the gut fluid system, whereby only NaTC formedmicelles. Oleic acid was added to the other three treatments to achievethe respective micelle-forming surfactant concentrations. This mimicsthe dynamic behavior of the main experiment in which more fatty acidswere produced over time as lipid digestion occurred. The differentmass concentrations of mixed micelles in this study had a significantinfluence on the micelle–water partition coefficient  = 81.86, p < 0.001 (SI Table S4). The post hoc pairwise comparisonsrevealed that all treatments differed from each other except the twolowest mass concentration treatment groups (5.7 and 6.9 g/L). However,the differences of the micelle–water partition coefficientswere generally less than 1 order of magnitude for the less hydrophobicPCBs. Since the variability of the log Kmicelles was less than 1 log unit, an average linear relationshipbetween log Kmicelle and log KOW with a slope of 1 across all mixed micelleconcentrations was determined (log Kmicelle = 0.99 (±0.03) × log KOW + 0.61 (±0.22); r2 = 0.86). Our linear regression was significantlydifferent from the relationship established by Schacht et al., 201670 , as shown by the solid green line in 73Due to the dynamic nature of the human digestive system, the concentrationsof lipids and micelles change over time as lipids break down. Sincethe FFA molecules produced during the hydrolysis process would formmicelles in the gut fluid,fficient 2A after F = 29.60, p < 0.001 .However, the regression coefficients of each treatment group alsodid not differ by more than 1 log unit. The limited differences inthe sorption partition coefficients for PCBs in the olive oil phaseat different concentrations (1.3–12.6 g/L) suggest that therewas no significant change in the sorption behavior at different oilconcentrations. Therefore, an average log–linear relationshipwas defined for all three olive oil concentration levels (log Koil = 0.85 (±0.04) × log KOW + 2.21 (±0.24); r2 = 0.84). In general, Koil valueswere significantly higher than KOW valuesby an average factor of 16 . Therefore, the micelle compartment has a higher sorption partitioncoefficient and affinity than LDPE but lower than olive oil (Koil > Kmicelles > KLDPE).The mixedmicelles have lower partition coefficients than oliveoil. Yet, they are higher than the LDPE-water distribution coefficients,which are determined from the transfer rate kinetics in this study though the trends were indistinguishablefor the high enzyme treatment and the denatured enzyme treatments. This supports our earlier suggestionthat hydrolysis may have continued to occur after the emulsion sampleswere extracted since lipase was still present in the emulsion.Lipid digestionwas demonstrated by the increase in the number of hydrogen protonsliberated over time with the release of FFA 1. MeasurkFFA, and maximum FFA limit, [FFAmax], are shown in The release of FFA in the high enzyme treatment differed significantlyfrom that in the low enzyme treatment 1. Both d32 The first-order rate constant (kFFA) in the low enzyme treatment is about 15 times higher than the kFFA for the high enzyme treatment, despite thelower enzyme concentration due to the denaturing process. This iscontrary to the generally confirmed theory that enzyme rate kineticswould increase with an increasing enzyme concentration. We argue thatthe fitted rate constant for the low enzymatic activity does not wellrepresent the actual enzyme kinetics due to the lack of data withinthe first 2 h of the reaction. This is also further supported by theless statistically significant kFFA inthe low enzyme activity treatment (p = 0.029) ascompared to the rate constant in the high enzyme activity. The [FFAmax] was 9 times higher for the high enzyme treatment thanthat for the low enzyme treatment. This significant difference impliesthat 9 times more FFAs were liberated for the high enzyme treatment.It is also important to note that the digestion process may be affectedby the concentration of MPs in the gut fluid. A recent study observedthat lipid digestion decreased with an increasing polystyrene MP concentrationas the particles heteroaggregated with lipid droplets and also actedas a sorbent for lipase, altering its secondary structure.32 Therefore, the enzyme digestion parameters fittedhere apply only to the digestive conditions of the present study.In the first 2 h of the digestion process, the initialrates ofFFA released were not distinguishable between the high and low enzymetreatments. However, both enzyme treatments showed a rapid increasein FFA initially, which agrees with an earlier study.74 Second, the FFA molecules produced from the hydrolysisprocess would form micelles in the gut fluid and thus increase thetotal micelle concentration for solubilization of PCBs.74 We observed a substantial increasein the PCB concentrations in LDPE over time during the lipid digestionprocess (S4). This indicates that PCBs sorbed in our foodproxy, i.e., the olive oil triglyceride fraction, were released duringlipid digestion and repartitioned in the gut fluid system, partiallysorbing onto the LDPE strips. In other words, this demonstrates thatMP particles can sorb chemicals from contaminated food during digestion.Here, the percentage increase in the PCBs bounded to the LDPE wasnot significantly different between the high and low enzyme treatmentswithin 72 h . This is becauseLDPE represented less than 10% of the main gut fluid components  in terms of mass concentration, whicheven reduces over time due to the removal of plastic strips for PCBmeasurement, whereas olive oil made up 80%. Furthermore, as discussedearlier, the LDPE in this study has a sorption partition coefficientlower than that of micelles and olive oil. As the oil breaks downand releases the sorbed contaminants, the micelles compete with LDPEfor the uptake of PCBs. Hence, we observed only a small differenceof about 30–80% in the concentration of PCBs  taken up by the polymer between the high and low enzyme treatmentsin the experimental setup .However, in nature, microplastic mainly remains in the gut duringdigestion, as it cannot be easily absorbed. Therefore, we used themodel parameters based on the experiment conditions to simulate theenvironmentally realistic exposure scenario that plastic strips werenot removed from the systems .We investigated an environmentally relevant scenario of the humandigestive tract; a human has bioaccumulated contaminants and ingestscontaminated food and contaminated plastic.21 However, in the present study,this biphasic behavior was not apparent due to the shorter time scale(SI formore details) were fitted to the high and low enzyme treatments (Table S8). For the control treatment, only PCB52 and PCB 101 showed a slight increase in concentration in LDPE overtime. The levels of contamination were close to the minimum detectionlimit and similar to the extraction method blank (Table S1 in the SI). Therefore, this contamination was not accountedfor in the respective high and low enzyme treatments as the levelof contamination was low, and the data still fitted well with themodel and initial boundary conditions set by the measured PCB concentrationsin the POM strip. PCB77 and 169 were omitted from further analysisdue to high relative standard deviations in the response factors ofreplicate measurements of calibration standards.As mentioned, the experiment was performed for alonger period than the standard upper gastrointestinal tract retentiontime. The PCB concentrations increased rapidly for the first 10 hand then approached a pseudoequilibrium state within 72 h. Previously,we observed biphasic chemical transfer kinetics during a 28 day exposureperiod.me scale3. Therefk1) were statisticallysignificant for all PCBs in both enzyme treatments,except PCB 209 (Table S8). They rangedfrom 236 to 1.37 × 107 h–1 and 138to 3.77 × 104 h–1 for high and lowenzyme treatments, respectively. The fitted k1 values for some congeners here were about 2−8 timeshigher than those from a similar chemical exchange kinetics experimentinvolving MP and food but without food digestion kinetics, in whichLDPE strips from the same source were used.21 The log k1 values were significantlypositively correlated with log KOW in both high (log k1 = 1.04 (±0.11) × log KOW − 3.01 (±0.73), r2 = 0.87, n = 10) and low (log k1 = 1.21 (±0.08) × log KOW − 4.28 (±0.51), r2 = 0.96, n = 10) enzyme treatments  = 5.19, p = 0.04 (SI Table S10). This may be dueto the higher amount of FFAs released in the high enzyme treatmentthan the low enzyme treatment, which may also facilitate the uptakeof PCBs onto the LDPE.78The fast sorptionrate constants , −82, k1) 21 The desorptionrate constants (k2) of the fast fractionwere statistically significant for all PCBs in both treatments exceptPCB 209 in the high enzyme treatment (Table S8). In the high enzyme treatment, k2 rangedfrom 0.283 to 4.36 h–1, translating to desorptionhalf-lives of 0.16–2.45 h. On the other hand, fitted k2 values in the low enzyme treatment were significantlylower than the high enzyme treatment, F = 13.82, p < 0.01 (SI Table S10), rangingfrom 0.087 to 0.289 h–1, translating to desorptionhalf-lives of 2.40–7.97 h. Furthermore, they were in a similarrange to the estimated k2 values fromour aforementioned study, which investigated the chemical kineticsof plastic with an inert food component.21 Unlike the uptake rate constants (k1), the desorption rate constants (k2)for the high and low treatments had weak positive correlations withhydrophobicity (Table S9), suggesting thatthe desorption process was not influenced by the bulk size or hydrophobicityof the PCB congeners. Both the uptake and desorption rate constantswere influenced by the enzyme digestion process. The high enzyme treatmentresults in a higher formation rate of FFA molecules, which may bedriving the PCB transfer in/out of the LDPE via facilitated transport.Here, facilitated transport refers to the additional transport ofchemicals between the aqueous phase and the LDPE due to the presenceof organic molecules in the diffusive boundary layer around the LDPE.30The reverse transfer kinetic (desorption) was alsoincluded inour model since chemical transfer occurs in both directions simultaneouslyas demonstrated earlier.KP)were derived from the fitted k1 and k2 estimates . The log KP positivelycorrelated with log KOW (log KP = 1.04 (±0.03) × log KOW + 0.05 (±0.23); r2 = 0.98, n = 20) with a slope close to 1,corroborating previous studies.86LDPE distribution coefficients . This implies that MP particles leaving the bodywould have substantially higher chemical concentrations than whenentering the body, which may seem counterintuitive yet logical basedon the processes at play. This increase, however, still implies adecrease in chemical bioavailability by about 0.11–0.87%  of the total chemical amount presentin the gut. This low percentage is mainly due to the removal of plasticstrips during the experiment, which significantly reduces the amountof PCBs taken up by the plastic phase. As mentioned earlier, in reality,ingested microplastics are typically not absorbed by the gut and thusremain in the gut during digestion.29 Weused the kinetic parameters based on the empirical data to simulatethe percentage reduction in bioavailability for the environmentallyrealistic scenario that the plastic remains in the gut over time . The results show that LDPE can reducebioavailability by 3–40% as lipids are digested. Furthermore,the percentage distribution of PCBs in the LDPE phase increases byabout 3–10% after 72 h of lipase digestion . This implies that fewer PCBs are absorbed bythe body via the FFAs and micelles as LDPE takes up a higher fractionof the PCBs released by the lipids during digestion.Afterthe lipid digestion was initiated for 72 h, LDPE took up part of thePCBs released from the digested olive oil triglycerides, resultingin an up to 75% increase in PCB concentrations in the LDPE . The overall reduction inchemical bioavailability is limited by the digestion rate kinetics,which is slower than the uptake kinetics (k1). Furthermore, LDPE is competing with micelles, which have about1.5–2 times stronger binding affinity than LDPE across thelog KOW range of PCBs tested inthis study.We alsoobserved a noticeable increase in the percentage reductionin the chemical bioavailability for more hydrophobic compounds  andcontaminated organic matter as food demonstrated that it is possiblefor MPs to clean the gut up to almost 90% without digestion if theMPs remain in the gut for more than 28 days and are eventually excretedout in the feces.p-dioxin (TCDD)and reduce TCDD concentrations.92Additionally,despite the difference in the exchange kinetics,the overall change in chemical bioavailability did not differ significantlybetween the high and low enzyme treatments. This implies that thedigestion dynamics will not result in significant differences in thechemical bioavailability when the plastic-to-food ratio in the gutis similar to that in the present study and also equally contaminated(at near-equilibrium). Therefore, here we show that a contaminatedLDPE is still capable of further taking up part of the released PCBsfrom food during digestion but with little effect on the digestiondynamics. Furthermore, the speculated facilitated transport attributedto the surrounding oil and free fatty acids enhances the partitioningof PCBs on LDPE, potentially making it a relevant chemical “cleaning”agent for ingested contaminated food. This similar “cleaning”phenomenon had been demonstrated earlier with olestra, a nondigestiblelipophilic dietary fat (similar to the LDPE here), which could takeup 2, 3, 7, 8-tetrachlorodibenzo-in vitro experimental design anda chemical kinetic model to investigate the chemical dynamics of MPsunder simulated gut conditions, including intestinal digestion processes.The model enables the accurate parameterization of kinetic parametersto describe the chemical transfer with MPs and digestion dynamics,which is crucial given the growing concerns regarding the chemicalimpacts of MPs. Our experimental setup replicated a scenario whereaquatic organisms or the human gastrointestinal tracts (GITs) areexposed to contaminated food and plastic through ingestion. We acknowledgethat other scenarios, such as contaminated food and clean plasticor clean food and contaminated plastic, have been previously discussedin the literature.93 Importantly, our chemical kinetic parameterscan also be applied to model the chemical behavior of HOCs with LDPEin other gut scenarios, such as different chemical concentrations,gut transit times, or variations in MP, lipid, micelle, and food massconcentrations. These results can be extrapolated to assess the exposureand risks of plastic-associated HOCs.93We presented an 96). We used lipids from olive oil as a proxy for food and LDPE asa proxy for MPs at specific concentrations. Further studies are neededto investigate other types of food and different ratios of MP to foodconcentrations since it has been demonstrated that MPs can also hinderdigestion.32We acknowledgethat our experimental study did not account for other dynamic changessuch as the absorption of micelles and their contents, such as short-chainfatty acids, across the gut lumen, as this was not a primary focusof our study. However, the chemical kinetic model framework and conceptualfindings from our study are also applicable to other dynamic gut systemsfor aquatic organisms and, thus, aquatic food webs. This is becausesodium taurocholate, a vertebrate bile salt used in our study to representmicelles in the gut, has been widely used to mimic intestinal fluidsof aquatic species  during digestion, increased the chemical fugacityin the gut. This increase serves as the driving force for biomagnificationin nature. In aquatic exposures, for instance, in the absence of microplasticingestion, it is realistic to consider biomagnification reaching asteady state for fish or invertebrate species native to the contaminatedaquatic environment. These organisms are already in chemical equilibrium(or at steady state) from the egg or larval stage even before theystart ingesting microplastic particles. In this scenario, when MPsare ingested, the increased fugacity in the digestive tract availablefor biomagnification leads to uptake by the ingested microplasticparticles as well see 3, thus a97 Insuch cases, the increase in sorbed chemical concentrations in MPscould result in a particle-mediated transfer. However, this is onlyrelevant to the very smallest particles , whichrepresent a marginal weight fraction of the entire ingested MP sizecontinuum.98 Furthermore, these particlesare assumed to have a very low absorption efficiency of approximately0.3% based on empirical data.9 Calculationshows that the mass of chemicals transported via this pathway is minimalcompared to the mass of chemicals absorbed from the gut by larger(>10 μm) particles due to the digestion of food, which aresubsequentlyegested . Thus, the net effect is that MPs remove HOCs from the biota underthe environmentally relevant scenario studied.The above discussion pertainsto the fraction of MP particles thatare too large to pass through the gut lining and be absorbed by thebody. It has been suggested that a fraction of contaminated MPs maybe small enough to pass through the gut lumen.As mentionedearlier, the conclusions of our study are applicableonly to scenarios whereby MPs are more or less equally contaminatedas food. It is important to highlight that if MPs are more highlycontaminated than food , thermodynamic principlessuggest that the chemicals would transfer to the lower fugacity compartmentof the gut. Consequently, in such a scenario, MPs would not reducethe chemical bioavailability during the digestion process.


As the beef industry continues to become more efficient and profitable, structural soundness will be important for the longevity of forage-based beef cattle. This study characterized the effects of age, sex, and sire line on mean foot angle and claw set scores of registered Angus cattle in Montana, USA. In our study, yearling bulls had greater mean foot angle and claw set scores than yearling heifers. Likewise, a greater proportion of yearling bulls had foot scores that were not ideal compared to yearling heifers. In addition, mean foot angle and claw set scores increased with increasing cow age, as well as the proportion of cattle with nonideal foot scores. The location of the combined worst foot was also influenced by cow age, with yearling cattle having predominantly front foot issues whereas mature cows often experienced hind foot issues. These data could potentially be used to improve the current American Angus Association (AAA) models and protocols for foot scoring.p < 0.01). Foot angle and claw set scores increased (p < 0.01) with advancing cow age. The combined worst foot changed quadratically with age (p < 0.01) with the majority of problem feet in cows aged 2 to 3 years and older being hind feet issues. The proportion of foot angle and claw set scores not equal to 5 increased quadratically with age (p < 0.01), with heifers having the lowest proportion of scores not equal to 5  compared to cows aged 4 years and older. Sire lines had an effect on progeny claw set (p < 0.05) and foot angle scores (p < 0.05), as well as variation of progeny foot scores. These data could potentially be used to refine expected progeny difference models.The objective of this study was to characterize foot angle and claw set scores of Montana’s (USA) registered Angus cattle using a total of 4723 cattle scored: 1475 yearling bulls, 992 yearling heifers, 1044 2- and 3-year-old cows, and 1212 cows ≥ 4 years old. Yearling bulls had a 0.12 and 0.20 greater mean foot angle and claw set score, respectively, compared to yearling heifers ( Proper foot conformation is important for productivity, health, and welfare in beef cattle production systems ,2. CorreMany factors likely affect foot conformation such as current production expectations, size and stature, age ,15,16, nIn response to concerns regarding feet and leg conformation, the AAA foot angle and claw set expected progeny differences (EPD) were developed in 2019 . These aFrom November 2021 to April 2022 (5 months), we used the AAA Foot Scoring Guidelines to subjectively score claw set and foot angle on 12 Montana registered Angus herds of various sizes . Over thThe AAA foot scoring guideline system  uses a nThe AAA foot angle and claw set expected progeny differences (EPD) were developed in 2019 . CurrentDue to the relative newness of collecting claw set and foot angle scores in Angus cattle, a primary goal of this research project was to characterize foot angle and claw set scores of registered Angus cattle in Montana. All cattle scored in this study were classified for age as either yearling bulls, yearling heifers, 2- to 3-year-old cows, or cows aged 4 years and older to evaluate the effects of sex and age on foot angle and claw set scores . Foot angle and claw set data were then analyzed using generalized linear models in an ANOVA framework including either sex or cow age classification as a fixed effect. For cattle that had foot angle and/or claw set scores differing from the ideal score of 5, we evaluated whether sex or cow age influenced the probability of the combined worst foot being a front or hind foot issue using generalized linear models following a binomial distribution in an ANOVA framework, with either sex or cow age classification as a fixed effect. Preplanned orthogonal polynomial contrasts were used to determine linear and quadratic effects of cow age classification on foot angle and claw set scores. Additionally, all sire lines within the dataset that had ≥75 offspring scored  were used to characterize the effect of sire line on progeny foot angle and claw set scores. Progeny foot angle and claw set data were analyzed using generalized linear models in an ANOVA framework including sire line as a fixed effect. All data were plotted and natural log-transformed if needed to satisfy assumptions of normality and homogeneity of variance. Significance was determined at an alpha of <0.05 with a tendency discussed with alpha <0.10. All statistical analyses were performed in R .p < 0.01). Yearling bulls had 1.74 times the number of individuals with a foot angle score different from 5 compared to yearling heifers . Yearling bulls and heifers were more likely to have front foot scores deviating from 5; however, yearling bulls had a greater proportion of front foot scores deviating from 5 compared yearling heifers (p < 0.01). Yearling bulls had a mean claw set score that was 0.2 points higher than yearling heifers (p < 0.01). Both yearling bulls and heifers were more inclined to have front foot scores that differed from 5. Yearling bulls exhibited a higher proportion of deviated claw set scores on the front foot compared to heifers  with heifers being the lowest, cows aged 4 years and older being the highest, and 2- and 3-year-olds being intermediate. For cows that did not score a 5 for foot angle, the proportion of scores on the front versus hind foot was quadratically (p < 0.01) impacted by age class. Specifically, heifers had a higher proportion of front foot scores that deviated from 5. In contrast, 2- and 3-year-old cows and cows aged 4 years and older were observed to have a higher proportion of hind feet scores deviating from 5, with cows aged 4 years and older having the highest proportion of hind foot score deviation.Comparisons were made across three different age groups of female cattle–yearling heifers (n = 992), 2- and 3-year-old cows (n = 1044), and 4-year-old cows and older (n = 1212). Foot angle scores increased linearly  with heifers being the lowest and cows aged 4 years and older being the highest. For cows not scoring a 5 for claw set, the proportion of scores on the front versus hind foot was quadratically  and is similar to sires 5, 6, 8, and 9 (p < 0.05). Sire 4 had a greater foot angle than sires 1, 3, 5, 6, 7, 8, and 9 (p < 0.05). Sire 7 exhibited the most ideal mean foot angle score in his progeny with sire 4 exhibiting the least ideal mean foot angle scores. Sires 1, 2, and 4 had greater mean claw set scores in their progeny versus sires 3, 5, 7, 8, and 9 (p < 0.05). Generally, the coefficient of variation will decline with an increase in the number of foot score observations. Sire EPDs and the coefficient of variation of foot angle and claw set scores are listed in p < 0.01; A descriptive statistical analysis of the nine sires with at least 75 progeny was performed to determine the mean claw set and foot angle, the range of claw set and foot angle scores, and the coefficient of variance for each sire . For fooChanges in foot scores, in this study, show an effect on the claw set and foot angle scores associated with age, sex, and sire line. The relative difference in foot angle and claw set scores represent significant differences in foot and leg conformation. Most animals scored using the AAA guidelines fall into foot angle and claw set scores of 4–8, and the scoring system uses only whole numbers. Age at scoring is fit (co)variable and sex is part of the contemporary group definition for the genetic evaluation model used to predict foot angle and claw set EPDs . EnvironAdvancing age influences foot angle and claw set. The effects of advancing age on foot conformation encompasses a variety of compounding stresses including pregnancies, lactations, wear and tear by everyday locomotion, and of course, the physiological effects of advancing age ,25. FurtIn addition, a significant transition of the scored foot from a front foot to a hind foot was observed with increased age. The analysis of which foot was scored when the score for each trait differed from score of 5 yielded a shift from the front foot to the hind foot, specifically in heifers in the 2- and 3-year-old age classes, bringing into question the most appropriate age at which female cattle should be scored. The question then becomes: do problem feet in yearlings translate to problem feet in older age cattle? Females of various age classes in this study were scored once. Perhaps future research could evaluate the same females year on year to observe whether the patterns reported in this study continue to exist, to further investigate the correlations and characterize the change from younger to older age cattle. The current recommendation of the AAA is to score registered female cattle annually .Montana Angus producers register and sell a significant number of bulls: 18% of all registered Angus bulls sold in 2022 were from Montana . Most daDifferences in management strategies and environment between the sexes could influence the claw set or foot angle. Physiological differences of bulls may result in increased body mass putting added pressure on front feet during the growing process. In contrast, the management of bulls versus heifers from weaning to yearling differs for most seedstock producers. Specifically, heifers are often managed very differently than their male counterparts, more often being allowed a slower and less intense growing period prepuberty. Heifers tend to more often be developed on pasture or in a feedlot, on a low-concentrate ration . Many reThe characterization of mean foot scores of progeny from sires with at least 75 offspring presented interesting and useful information for comparing sires. As presented in R = 0.34, p < 0.01). Wang [For the entire data set, which included both mature and yearling female cattle, foot angle and claw scores were compared and found to be moderately correlated phenotypically (1). Wang  found th1). Wang ,21. This1). Wang .Our study will aid in the continued refinement of the foot scoring protocol. The frequency of hind versus front hoof selection is significantly influenced by age, with yearlings having predominantly front foot problems, whereas 2-year-old cows and older have predominantly hind foot problems. Additional improvements may be possible with continued model refinement and improvements with scoring guidelines specific to age and sex effects, such as enacting an age in which scoring is conducted or specifying whether a front or hind foot should be scored at a given age. The AAA guidelines have been beneficial in developing selection tools for optimal conformation. However, as with all tools, further refinement of models and sampling protocols are necessary to continue to grow the adoption of foot scoring protocols and genetic selection of beef cattle.Beef breed associations and beef cattle breeders are emphasizing selection pressure on structural soundness. This increased need is likely due to the production demands placed on beef animals over centuries of cattle breeding. Ideal structural soundness often equates to better locomotion, which, in turn, increases an animal’s productivity and longevity in numerous production scenarios ,5,6. As Perhaps in the future, this subjective approach will lead to a more quantitative method with the use of technology . Foot scoring guidelines provided by the AAA for their members allow the opportunity to continue to improve the selection tools for Angus cattle for foot angle and claw shape. The success of this EPD will depend upon acceptance by commercial cattlemen as a selection tool and the continued collection of foot scores by breeders.