Our data revealed that viability and functionality of F. mosseae and R. irregulare extraradical hyphae were uncoupled from host plant lifespan. Indeed, ERM spreading from roots of intact or shootless plants showed comparable levels of viability, similar structural traits and ability to establish mycorrhizal symbioses with new plants, as long as five months after shoot removal. Our findings expand the current knowledge on AMF biology and life cycle, providing data on ERM long-term survival in the soil of two Glomeracean species, functional to the prompt establishment of mycorrhizal symbioses and to the maintenance of soil biological fertility.Arbuscular mycorrhizal fungi (AMF) are obligate symbionts, living in associations with the roots of most land plants. AMF produce wide networks of extraradical mycelium (ERM) of indeterminate length, spreading from host roots into the surrounding soil and establishing belowground interconnections among plants belonging to the same or to different taxa. Whether their lifespan and functionality are limited by host plant viability or can be extended beyond this limit is unknown. To address this issue, we performed time-course studies to investigate viability and functionality of ERM produced in an  Many works support the obligate biotrophy of AMF for carbohydrates and their inability to synthesize fatty acids, suggesting that such carbon sources may play an important role in regulating AMF intraradical proliferation, arbuscule development and life cycle completion2. Belowground mycorrhizal networks extend the absorbing surface area (up to 40 times) growing in every direction4, efficiently exploring the soil and increasing plant uptake of phosphorus, nitrogen, sulphur, immobile micronutrients such as copper and zinc and other soil-derived mineral cations9. In addition, AMF protect plants from biotic and abiotic stresses, such as pathogens, drought and salinity13 and affect plant secondary metabolism, enhancing the synthesis of beneficial phytochemicals, thus contributing to the sustainable production of high-quality food15.Arbuscular mycorrhizal fungi  are beneficial soil symbionts establishing mutualistic associations with the roots of 80% of plant species and the large majority of food crops, including cereals, legumes, vegetables and fruit trees. AMF are key elements of soil fertility, which depends on biological, chemical and physical components, of plant nutrition and productivity, absorbing soil mineral nutrients by a fine network of extraradical hyphae growing from colonised roots into the soil17. Other studies investigated the extent and interconnectedness of AMF hyphal networks, which represent critical factors for the maintenance of nutrient flow from the extraradical to the intraradical phase. ERM density is estimated to range from 2.7 to 20.5 m/g of soil19, with a mean growth rate of 0.74–1.1 m d−1 and a specific weight of 3.8–7.8 μg m−1 20. ERM formed by members of the family Glomeraceae, the most abundant AMF in agricultural soils, are highly interconnected by means of fusions (anastomoses) between contacting hyphae, reaching the value of 100–410 anastomoses per gram of soil21. Such structural traits are of functional significance for the uptake, translocation and flow of nutrients from soil to host roots, as ERM extent and interconnectedness have been shown to be positively correlated with host growth response variables and P content, affecting symbiotic performance and plant growth and nutrition22.Several molecular studies investigated the functioning of extraradical mycelial networks (ERM) and revealed that genes encoding proteins for transport of mineral nutrients, such as phosphorus, zinc, nitrogen, are differentially expressed in ERM hyphae, thus confirming their key role in mineral uptake in the soil-fungus interface25. The role of plant root cell death in determining the viability of AM fungal symbionts has been long debated, and previous studies showed that, beyond spores, both living and dead root fragments can represent a source of inoculum for newly developing roots28. Notwithstanding, the question on the viability and lifespan of extraradical fungal hyphae after the detachment of root system from the host plant shoot has remained long unanswered.So far, ERM ability to survive and maintain infectivity independently from the host plant lifespan have not been adequately investigated, although the fate and behaviour of dead colonized roots and their connected extraradical hyphae after host plant harvest may affect the production of spores, fundamental for the survival of AMF communities in the soil. The conservation of the whole soil mycorrhizal potential, represented by colonized roots, ERM and spores, represent an essential factor of soil biological fertility in organic and sustainable food production systemsin vivo whole-plant experimental system and two worldwide distributed Glomeracean AMF, Funneliformis mosseae and Rhizoglomus irregulare, were utilised. On a time-course basis, after removing the shoots of the host plants, we monitored for 5 months (i) ERM structural traits and viability by assessing cellular succinate dehydrogenase activity, (ii) ERM maintenance of functionality by testing its ability to establish mycorrhizal symbioses in young seedlings placed in contact with ERM of different ages.The aim of the present work was to investigate ERM viability and functionality in shootless mycorrhizal plants, after assessing viability loss of roots. To this aim, an C. intybus showed viable length rates of 55 ± 3% and 30 ± 2%, 24 h and 48 h after shoot removal, respectively. On the third day, 14 ± 1% viable length was detected, mainly at the root tips, whereas on the fourth day all the roots were not viable .After FDA staining carried out in Experiment 1, roots of shootless At each time point of Experiment 2 (Exp. 2), SDH localisation did not reveal viable cells in root systems of shootless plants Fig. , althoug2, and mycelial lengths were similar among the two AMF tested   extraradical hyphae was continuous for the whole duration of the experiment (150 d) in both intact and shootless plants, and showed significant differences among the different time points (Table R. irregulare IMA6 (hereafter IMA6), respectively. The extent of viable ERM in IMA1 mirrored the trend showed by total length, while in IMA6 it did not change with time , ERM maintained its structural traits. Histochemical localisation of SDH activity allowed the detection of protoplasmic continuity and viable anastomoses in hyphae growing from roots of both intact and shootless plants, ranging from 32 to 44% of total contacts, with no differences in interconnection rates among shootless/intact plants and fungal symbionts. ERM hyphae connected to dead roots maintained their viability and showed SDH activity for 5 months after shoot removal Fig. .11,24 = 46.3, P < 0.001)  and intact plants   compared with the near-root zone , whereas the correlation was low or not significant for data obtained from ERM produced by intact plants  or distant from them (1 cm from the ERM edge), while a dramatic viability reduction over time was found in ERM regions distant from roots of shootless plants Fig. . In thes2 = 0.92, P < 0.001)  were significantly different from those detected at 90, 120 and 150 d time points (ranging from 34.5 to 61.2%)   Table .Table 2PThe data obtained in this work revealed that the cellular viability and functionality of extraradical hyphae are uncoupled from the host plant lifespan in two Glomeracean AMF isolates. For the first time, we showed that, after shoot removal, the growth of ERM spreading from detached root systems was comparable with that from intact plants, and continuous for the whole duration of the experiment, 150 d. Moreover, ERM showed comparable and high levels of viability, and maintained its ability to establish mycorrhizal symbioses with new plants, as long as 5 months after shoot removal. These findings expand the current basic knowledge on AMF biology and life cycle and on ERM role in the maintenance of mycorrhizal potential in the soil.29, in agreement with data reporting that in Lolium perenne roots detached from the shoots lost up to 60% of their initial N and up to 70% of their P within the first three weeks30. Previous works investigated the impact of plant shoot removal on ERM nutrient transfer activity in plant interconnected by AMF extraradical hyphae and showed significant transfer of N and P from dying donor roots to receiver plants after shoot removal from the donor plant32. Accordingly, Müller et al.33 found significant translocation of 15N from dead donor roots to receiver plants in mycorrhizal tomato. Such findings represent evidence of the persistence of ERM viability and functionality in dead root systems, recorded over short periods of time, 2 and 6 weeks, respectively. As our work lasted 5 months, we could suppose that AMF hyphae were able to recapture from detached roots not only nutrients early released after shoot removal, and not only N and P, but also all the other nutrients they need for the continuous growth detected here. Indeed, arbuscules, the main sites where nutrient exchanges between the two partners of AM symbiosis occur, were viable in dead roots 5 months after shoot removal, suggesting that they might be still able to uptake nutrients from host roots and to support ERM life, thus leading to the maintenance of soil mycorrhizal inoculum potential after crop harvest  or gave inconsistent results  when inoculum was represented by dead roots, suggesting a differential behaviour among AMF, possibly related with their sensitivity to disturbance44.34, while soil mycorrhizal potential and diversity was enhanced by mycotrophic cover crops46, as the result of the development of extensive mycelial networks, retaining their viability and infectivity after harvest. On the other hand we observed a similar decrease in average viability in ERM connected to both intact and shootless plants at 150 days of growth: it would be interesting to investigate whether ERM reduced viability over time, even when connected to intact plants, due to changes in plant nutrient transfer ability occurring in aged hosts.Hyphal ability to survive, grow and retain host infection capability independently from the host plant lifespan may represent an important trait, functional to the conservation and increase of mycorrhizal potential of soils. Indeed, ERM produced by native AMF was able to survive Mediterranean summer dry conditions, maintaining its colonisation abilityIn conclusion, our findings offer a new vision of AMF life cycle in the soil, where fungal extraradical mycelium, growing from both living and dead roots, represents a long-term survival structure - beyond quiescent spores and mycorrhizal root fragments - functional to the prompt establishment of mycorrhizal symbioses and to the maintenance of soil biological fertility and mycorrhizal inoculum potential in agricultural soils.in vivo experimental design was devised in order to obtain intact AMF extraradical hyphae developing from mycorrhizal roots, to be monitored after the detachment of host plant shoots47. Time-course experiments allowed the study of lifespan and structural traits of ERM connected to roots of shootless plants, as well as its colonisation ability . A schematic outline of the experimental design is presented in Supplementary Figure Cichorium intybus seeds were surface-sterilised, germinated and grown for 10 days in sterile quartz grit and then inoculated with 50 mg (fresh weight) of sieved inoculum. Such inoculum, composed of spores or sporocarps, mycelium and colonised roots, was obtained from pot-culture soil of two different AMF, Funneliformis mosseae isolate IMA1 and Rhizoglomus irregulare isolate IMA6 , which was suspended in water and sieved (repeating this procedure up to 10 times) through a 100-µm-mesh size sieve. Inoculated plants were grown in 10-cm diameter pots filled with sterile quartz grit, in a growth chamber with 25 °C day and 21 °C night temperature, 16 h of light per days. After four weeks’ growth, grit was washed from roots, which were checked for the absence of contaminating fungal mycelium or propagules other than AMF, and spores and sporocarps adhering to plant roots, along with most extraradical hyphae, were carefully removed with forceps under a Leica M 205 C dissecting microscope  and root systems were singly wrapped in nylon nets (40 µm mesh)47. Each root system was then placed between two semicircular 13-cm diameter Millipore™ membranes (root sandwiches), transferred into 14-cm diameter Petri dishes containing moist sterile quartz grit, with the root-containing lower half of plates wrapped into aluminium foil, and maintained in the growth chamber47. After ten days, plants which showed 12–15 cm2 of the membrane areas covered by ERM (assessed with a transparent graph paper) were selected for the different experiments, and the shoots of plants of the “shootless plant” treatment were cut with sterile blades (1 cm over the upper edge of membrane sandwiches), without disturbing the root systems and the mycorrhizal extraradical networks growing on the membranes, while plants belonging to the “intact plant” treatment were not affected   in phosphate buffer 48. This staining is a cell-permeant esterase substrate that can serve as a viability probe indicating both enzymatic activity, which is required to activate the fluorescence, and cell-membrane integrity, which is required for intracellular retention of the fluorescent product48. The roots were stained for 10 min at 21 °C, rinsed in the same buffer and observed under the Reichert-Jung Polyvar epifluorescent microscope equipped with excitation filter BP 450–495, barrier filter LP 520 and dichroic mirror DS 510. The proportion of viable and total root length was assessed on three aliquots (each of 4 × 3 cm root length) for each root system by the gridline intersect method49, using a grid eyepiece at 100× magnification.To evaluate root viability maintenance after shoot detachment, 12 plants of F. mosseae IMA1 and R. irregulare IMA6 ERM connected to roots of shootless and intact plants were stained for the localisation of succinate dehydrogenase (SDH) activity50 followed by Trypan blue in lactic acid (0.05%). On each membrane, check of roots and intraradical fungal structures viability and assessment of viable and total ERM hyphal density were carried out under the dissecting microscope. ERM hyphal densities  were assessed on three replicate membranes at each time point and for each treatment, by the gridline intersect method, using a microscope eyepiece grid at 320 × magnification47. On each replicate membrane two different measures of both viable and total hyphal density were compared to examine ERM viability: (i) in six randomly selected areas of 9 mm2, (ii) in six areas of 9 mm2 within 1 cm from nylon-enclosed roots and in six areas selected within 1 cm from the edge of ERM. Moreover, the area explored by ERM was assessed by using a transparent 5-mm-square grid. Total and viable hyphal lengths were calculated from measured data, multiplying mean hyphal densities by the area explored by the mycelial network. Areas of membranes (10 × 25 mm each) covered by ERM were cut, mounted on microscope slides and examined under a Reichert-Jung  Polyvar microscope. ERM hyphal contacts were counted at x320 magnification on at least three samples from each replicate membrane and perfect fusions were recorded when hyphae anastomosed and protoplasm continuity could be verified by SDH staining.A total of 36 replicates (18 shootless plants and 18 control intact plants) for each AMF isolate were prepared and sequentially harvested 0, 30, 60, 90, 120 and 150 days after shoot removal . At each time point, membranes of sandwich systems containing C. intybus for each AMF isolate were obtained using the experimental system described above. Three sandwiches for each AMF were carefully opened at different time points  and two C. intybus seedlings were inserted in the sandwich, placing their roots on ERM spreading from the roots  and three and two-ways ANOVA were performed to assess the significance of the different factors involved  on fungal parameters (viability rates and length of ERM) and on plant root colonisation. Percentage data were analysed after arcsin transformation. One-way ANOVA followed by Tukey’s HSD multiple range test (homogeneous variances) or Welch’s test (not-homogeneous variances) was used to detect significant differences among data obtained for each AMF isolate and plant treatment (different time points) or for each isolate and time point (different plant treatments). Correlation and regression analyses were carried out to reveal relationships among fungal parameters and time and between ERM viability and colonisation ability. All statistical analyses were performed with SPSS Statistics version 23 .The datasets analysed during the current study are available from the corresponding author on reasonable request.Supplementary file


Anemia in young children is a global health problem. Risk factors include poor nutrient intake and poor water quality, sanitation, or hygiene.We evaluated the effects of water quality, sanitation, handwashing, and nutrition interventions on micronutrient status and anemia among children in rural Kenya and Bangladesh.We nested substudies within 2 cluster-randomized controlled trials enrolling pregnant women and following their children for 2 y. These substudies included 4 groups: water, sanitation, and handwashing (WSH); nutrition (N), including lipid-based nutrient supplements  and infant and young child feeding (IYCF) counseling; WSH+N; and control. Hemoglobin and micronutrient biomarkers were measured after 2 y of intervention and compared between groups using generalized linear models with robust SEs.P = 0.102).In Kenya, 699 children were assessed at a mean ± SD age of 22.1 ± 1.8 mo, and in Bangladesh 1470 participants were measured at a mean ± SD age of 28.0 ± 1.9 mo. The control group anemia prevalences were 48.8% in Kenya and 17.4% in Bangladesh. There was a lower prevalence of anemia in the 2 N intervention groups in both Kenya  and Bangladesh . In both trials, the 2 N groups also had significantly lower prevalences of iron deficiency, iron deficiency anemia, and low vitamin B-12 and, in Kenya, a lower prevalence of folate and vitamin A deficiencies. In Bangladesh, the WSH group had a lower prevalence of anemia  than the control group, whereas in Kenya, the WSH+N group had a lower prevalence of anemia than did the N group , but this was not significant  and NCT01704105 (Kenya).These trials were registered at  Trichuris trichiura infection, can cause blood loss leading to anemia  arms in both trials, but none in the water, sanitation, and handwashing (WSH) arms (1) control; 2) N, comprising infant and young child feeding (IYCF) behavior change messaging plus lipid-based nutrient supplements (LNSs); 3) WSH; and 4) WSH+N. These substudies evaluated 2 hypotheses: 1) improvements in N, WSH, or WSH+N would improve biomarkers of micronutrient status and reduce the risk of anemia in young children and 2) the combination of WSH+N would improve biomarkers of micronutrient status and reduce the risk of anemia more than either WSH or N alone.The WASH Benefits Study evaluated the effects of water, sanitation, handwashing, and nutrition interventions delivered alone and in combination in 2 highly comparable trials in Kenya and Bangladesh . The primary outcomes of the trial included length-for-age Detailed descriptions of the 2 trials have been previously published .The trial was implemented in 3 counties in western Kenya—Kakamega, Bungoma, and Vihiga. Geographically matched village clusters were block-randomized into 1 of 8 study arms: chlorine treatment of drinking water (W); improved sanitation limiting exposure to feces (S); handwashing with soap (H); combined WSH; IYCF counseling plus small-quantity LNSs (N); combined WSH+N; active control in which children were visited by a health promoter monthly; and passive control with no intervention household visits.Villages were eligible for inclusion into the trial if they were rural, with most of the population reliant on unimproved sanitation facilities, without widespread access to chlorinated water, and without any active water, sanitation, handwashing, or nutrition programs. Households were eligible if there was a woman in her second or third trimester of pregnancy who planned to live in the community for ≥2 y and who could speak Kiswahili, Luhya, or English. Intervention clusters were formed by including a minimum of 6 eligible pregnant women residing in ≤3 neighboring villages. There were no buffer zones between clusters due to geographic constraints.Community health promoters (CHPs) were nominated by women in the community and trained to provide the intervention-specific behavior change activities as well as instructions for provision of supplements or hardware use. In both the active control and intervention arms, they were trained to measure midupper arm circumference to identify and provide referrals for potential cases of severe acute malnutrition.Each intervention package consisted of a comprehensive behavior change promotion program, including key messages; visual aids in the form of flip charts, posters, and reminder cue cards; interactive activities with songs, games, and pledges; and the distribution of arm-specific hardware, products, or supplements . CHPs weSupplemental Table 1. At the start of the trial, supplements were distributed by CHPs, whereas in the second year of the study, project staff provided the monthly rations. Supplements were provided for the index child plus any other age-eligible siblings in the household. Caregivers were instructed to mix one 10-g sachet into the child's complementary foods twice per day. Key messages focused on standard IYCF recommendations, including maternal dietary diversity during pregnancy and lactation, early initiation of breastfeeding, exclusive breastfeeding from age 0 to 6 mo and continued breastfeeding through 24 mo, timely introduction of complementary foods at 6 mo, dietary diversity, feeding frequency, and feeding during illness.In the N intervention arm, households received monthly rations of micronutrient-fortified, small-quantity LNSs (Nutriset) when children were between the ages of 6 and 24 mo. The composition of the LNSs in comparison to the Recommended Nutrient Intakes for children is shown in In the WSH intervention arm, CHPs advocated for a variety of behaviors to improve water quality, sanitation, and handwashing practices within the home. Specifically, they promoted treatment of drinking water with sodium hypochlorite using either chlorine dispensers installed at the point-of-collection in study villages or bottled chlorine provided directly to households. They also used chlorine test strips to spot-check household chlorine concentrations during monthly visits; results were used to improve counseling. Existing latrines were upgraded and improved by installing a plastic slab with a tight-fitting lid. Households with no latrine were provided with a new one. CHPs provided a “sani-scoop” with a paddle to remove animal and human fecal material from the yard surrounding the home and child potties for each child aged <3 y in the study compound. Households were also provided with 2 dual tippy-tap handwashing stations, one near the latrine and a second near the cooking area. Stations were designed to have 2 foot-pedal–operated jerry cans that could be tipped to dispense a small stream of either soapy water or rinse water . CHPs fiAdherence to LNS recommendations was high (>90%) in the N arms, but there were no differences in complementary feeding practices between groups , 15. AdhSupplemental Figure 1); and it had no major water, sanitation, or focused nutrition programs underway or planned. Clusters were randomized by geographic blocks into 1 of 7 study arms: chlorine treatment of drinking water (W); improved sanitation limiting exposure to feces (S); handwashing with soap (H); combined WSH; IYCF counseling plus small-quantity LNSs (N); combined WSH+N; or a control group that received no intervention. In contrast to Kenya, there was no active control arm.The WASH Benefits Bangladesh Trial was implemented in subdistricts of the Gazipur, Kishoreganj, Mymensingh, and Tangail districts. The study area was selected because it had low groundwater iron and arsenic according to data obtained from the Department of Public Health Engineering/British Geological Survey/Department for International Development National Hydrochemical Survey and a survey conducted before study initiation , 17  tablets (Medentech) to treat and safely store their drinking water. Households were encouraged to fill the vessel, add 1 tablet, and wait 30 min before drinking the water. Households that did not have access to an improved latrine with a slab and functional water seal in their compound were provided either a new latrine or improvements to an existing latrine. Households were also provided a scoop that could be used to clean the home environment and a child potty for all children aged <3 y for the safe disposal of child feces. Handwashing practices were promoted and supported by providing 2 handwashing stations, one placed near the latrine and the second placed near the kitchen. Each station contained a water reservoir with a tap, a basin to collect the rinse water, and a soapy water bottle. CHPs provided a regular supply of detergent for making the soapy water. The WSH+N intervention group received all of the aforementioned behavioral interventions, hardware, and products. The adherence to LNS supplementation and all behavioral recommendations was high across all groups throughout the trial  and dietInterventions were randomly assigned at the cluster level with the use of codes generated by an off-site investigator independent of the data collection team via a random-number generator. Groups of adjacent clusters were block-randomized into the 6 intervention arms or a double-sized passive control arm (in Bangladesh) or double-sized active control arm and single-sized passive control arm (in Kenya). Participants were informed of their group assignment after the baseline survey. Masking participants was not possible due to the nature of the interventions. Although data collectors were not directly informed of the random assignment, they may have inferred the intervention group through observation of materials within the home during subsequent household visits. Masked technicians completed the laboratory analyses.1) restless, irritable; 2) sunken eyes; 3) drinks eagerly, thirsty; and 4) pinched skin returns to normal position slowly or the child being listless or unable to perform their normal activities.In both trials, substudy clusters were selected from the N, WSH, WSH+N, and control arms (active in Kenya and passive in Bangladesh). Clusters were selected based on the logistical feasibility of the preservation and collection of biological specimens, as well as their transport to the central laboratory. The primary objective of the substudies was to assess biomarkers of environmental enteric dysfunction, which will be reported elsewhere. Index households with live-born infants residing in selected clusters were invited to participate in the substudy activities. Exclusion criteria included symptoms of moderate to severe dehydration on the day of the survey, as follows: In both trials, after consent and enrollment, enumerators administered a baseline questionnaire asking respondents to report on their household characteristics, animal and other asset ownership, food insecurity status using the Household Food Insecurity Access Scale  in BanglSubstudy visits were conducted within the home in Bangladesh and at a central site in the community in Kenya. For each child, a maximum of 7.7 mL blood was collected by a trained phlebotomist via venipuncture into a Sarstedt monovette serum collection tube and a Sarstedt monovette lithium heparin trace element–free plasma collection tube. Hemoglobin concentrations were measured in venous whole blood at the point of collection using a portable spectrophotometer (Hemocue 301) that was regularly checked using standardized quality-control procedures. In Kenya, malaria was tested using rapid diagnostic test kits  at the time of collection. In Kenya, blood samples were placed on ice packs, centrifuged at 3500 rpm for 15 min at ambient temperature within a mean of 1 h of collection, and then plasma and serum aliquots were placed on ice packs in a cooler where they remained for ∼2.5 h until they could be transferred to a −20°C freezer. Within ∼2 wk of collection, samples were subsequently transferred to −80°C freezers at the Kenya Medical Research Institute (KEMRI) in Nairobi for long-term storage. In Bangladesh, blood samples were placed on ice, transported to the project laboratory, centrifuged at 3500 rpm for 15 min at ambient temperature, and stored in a −80°C freezer. The mean time from blood collection to centrifugation was 8.5 h.Specimens were shipped on dry ice to collaborating laboratories for analysis. Serum retinol-binding protein (RBP), ferritin, soluble transferrin receptor (sTfR), α-1 acid glycoprotein (AGP), and C-reactive protein (CRP) were measured using ELISA methods  VitMin , 24 at Kn = 375 children/group, assuming 54 clusters/arm and 7 children/cluster (https://osf.io/qa43y/). Using this number, we estimated that we would be able to detect a mean difference in hemoglobin between groups of 3.4–3.9 g/L or a difference in the prevalence of anemia of 11.3–11.9 percentage points based on an assumed mean hemoglobin of 103 g/L and prevalence of anemia of 47–85%, based on previously published data from children in nearby study areas  between any intervention arm and the control arm assuming 80% power, a 2-sided type 1 error of 5%, and an intracluster correlation coefficient of 0.15, resulting in a sample size of dy areas , 26. Thedy areas .Mothers provided written informed consent for themselves and their infants. The study protocol was reviewed and approved by the Committee for the Protection of Human Subjects at the University of California, Berkeley, the Institutional Review Board at Stanford University, the Ethical Review Committee at the International Center for Diarrheal Disease Research, Bangladesh, and the Scientific and Ethics Review Committee at the KEMRI. Children with hemoglobin <70 g/L or who tested positive for malaria with fever or sickle cell disease (HbSS) were referred for treatment.https://osf.io/dsrv2). Analyses were independently replicated by 2 investigators (CDA and KAB) using R version 3.5.0  and Stata version 14 (StataCorp LLC). All investigators were blinded to group assignment until primary analyses were completed.Detailed analysis plans were prespecified and publicly posted  analysis that reweighted the analysis population to reflect the original enrolled population , 43. Then = 75), child death (n = 80), or not attending the study visit (n = 741). Among those who attended the study visit, an additional 608 refused to provide a blood sample and 142 had missing data due to insufficient blood volume collected or other reasons. Follow-up rates were similar across groups. Enrollment characteristics were generally balanced . The only notable difference was that those who were included in the analysis sample were less likely to be firstborn children (19.1%) compared with those in the substudy with missing data (26.7%) or those in the rest of the main study sample (24.0%). Adherence to LNS supplementation was high (>94% of expected sachets consumed) among age-eligible children at midline and endline .In the Kenya trial, households were enrolled between November 2012 and May 2014 and the substudy follow-up occurred from July 2015 to April 2016. A total of 190 clusters with 2304 households were included in the substudy . Included participants also had similar baseline characteristics to those not selected into the substudy. Just as in Kenya, the adherence to LNS supplementation was high (>90% of expected sachets consumed) at both time points of measurement among the age-eligible children .In the Bangladesh trial, study enrollment occurred between May 2012 and July 2013. A total of 267 clusters with 2039 compounds were included in the substudy, balanced across the 4 intervention arms  had higher concentrations of hemoglobin, RBP, ferritin, hepcidin, and vitamin B-12 and lower sTfR 3 and 4. Supplemental Table 6). There was a 9.0 percentage point lower prevalence of anemia in the WSH+N group compared with the N group, but this difference was not significant (P = 0.102). There were significantly lower prevalences of iron deficiency, iron deficiency anemia, vitamin A deficiency, and vitamin B-12 depletion or deficiency in both of the groups that received the N intervention package . Folate deficiency was nearly eliminated in those 2 groups, and there was a concurrent higher proportion of children with high folate concentrations.Compared with the control group, there was a 26% and 44% lower prevalence of anemia in the N and WSH+N groups, respectively . This coThere were no differences in mean hemoglobin or micronutrient status markers between the WSH and the control groups 3 and 4. Supplemental Table 7). Correction for inflammation reduced the estimated prevalence of vitamin A deficiency but slightly increased the prevalence of iron deficiency and iron deficiency anemia across all groups. Estimated differences between groups were similar to those calculated using the non–inflammation-corrected values.In the control group, the prevalence of malaria was 18.9% and inflammation as measured by CRP or AGP was present in 53.5%; these did not differ between study arms . There was no evidence of effect modification by age, sex, or household hunger score , although a small sample size of households with moderate to severe hunger limited our ability to detect a potential interaction. There was some evidence of effect modification with hemoglobinopathy trait, but there was not a consistent pattern in the stratified analysis .The results were similar after adjustment for covariates and in the IPCW sensitivity analyses . The WSH group did not differ from the control group in any of these biomarkers. RBP concentrations did not differ between groups. Folate concentrations were significantly lower in the WSH and WSH+N groups. There were no significant differences between the WSH+N group and the N group.In the 2 N intervention groups (N and WSH+N), hemoglobin, ferritin, hepcidin, and vitamin B-12 concentrations were significantly higher, whereas sTfR was significantly lower compared with the control group . Iron deficiency anemia was 74–87% lower and the prevalence almost eliminated (<3%) in these 2 groups. The prevalence of anemia was 26% lower in the WSH group compared with the control, but there was no added benefit of WSH+N over N alone. The prevalence of vitamin A deficiency did not differ between groups (Table 7). The prevalence of vitamin B-12 depletion or deficiency was significantly lower by 45% in the N group, but the magnitude of the reduction was attenuated and not statistically significant in the WSH+N group compared with the control group. There was a higher prevalence of folate deficiency in the WSH group compared with the control group and no difference in the prevalence of high folate between any of the groups.Similar to the results seen in the continuous concentration measures, the prevalences of anemia and iron deficiency were ∼50% lower in the 2 N groups compared with the control group . For aneSupplemental Figure 2). The other nutrient biomarkers did not show such a strong seasonal pattern. In models adjusted for covariates, including month of sample collection, mean folate concentrations did not differ between groups . All other adjusted group comparisons were similar to the unadjusted estimates.There was a strong seasonal pattern in mean folate concentrations and the timing of sample collection varied between groups . The results were also similar in the IPCW analyses . Specifically, there was a 75–86% higher median ferritin and 20–21% lower median sTfR in the N and WSH+N groups, compared with the control, among children aged ≤28 mo. In children aged >28 mo, there was a more modest 47–53% higher median ferritin and 5–7% lower median sTfR in those 2 groups compared with controls. There was also a significant interaction with food insecurity with regard to vitamin B-12 status: effects were apparent only among children in food-insecure households in the WSH, N, and WSH+N groups compared with controls and no effects were evident in children from food-secure households . There were no significant interactions with child sex or thalassemia trait .Correction for inflammation reduced the estimated prevalence of vitamin A deficiency, but slightly increased the prevalence of iron deficiency and iron deficiency anemia across all groups. Estimated differences between groups were similar to those calculated using the non–inflammation-corrected values . Iron deficiency anemia was nearly eliminated in the 2 N intervention groups in Bangladesh. There were also reductions in the prevalence of low vitamin B-12 status in both trials. The prevalences of vitamin A deficiency and folate deficiency were reduced in the N arms in Kenya, but not in Bangladesh. In Bangladesh, children in the WSH intervention group had a 26% lower prevalence of anemia compared with the control group, but there were no effects of the WSH intervention on any of the micronutrient biomarkers, and there was no added benefit of combined WSH+N as compared with the individual nutrition-specific intervention.In contrast, in Kenya, there was a 9 percentage point reduction in the prevalence of anemia in the combined WSH+N group compared with N alone, although this difference was not significant.Although these 2 trials are comparable in many ways, there are a number of notable differences in the design, implementation, and context. The prevalences of anemia and iron deficiency were substantially higher in Kenya than in Bangladesh and so the potential of benefit was greater in Kenya. The prevalence of anemia of 17.4% in Bangladesh was lower than expected. By contrast, the reported prevalence of anemia among preschool children in the 2013 Bangladesh National Micronutrient Status Survey was 33% . The difIn the Kenya study, the N intervention package also significantly reduced the prevalences of vitamin A deficiency and folate deficiency compared with controls, whereas this was not apparent in the Bangladesh study. One possible explanation for the variation in estimated impacts on micronutrient status is that there were delays in the timing of sample collection in the Bangladesh study due to security concerns arising from civil unrest, such that children were, on average, 4 mo beyond the age at which they stopped receiving LNSs. Indeed, effect sizes were larger among young children in stratified analyses in that trial. Serum folate is reflective of recent intake , so we mThere were somewhat contrasting results on the effects of the WSH intervention on anemia in the 2 trials. In Bangladesh, there was an effect of WSH alone, whereas in Kenya the prevalence of anemia was 9 percentage points lower in the WSH+N group when compared with the nutrition-specific intervention group (N). Although this was not significant in Kenya, the difference was of a large magnitude and suggests that there may be additional benefit from combining WSH with a nutrition intervention package. The lower than expected sample size due to a high rate of missing data reduced the statistical power to detect this difference. Using our empirical data of 36.2% and 27.3% prevalences, an intracluster correlation of 0.108, 41 clusters per arm, and 4 children per cluster, the study would have needed 569 children per group to have 80% power. By contrast, the study included only 205 children in the N group and 167 children in the WSH+N group.Trichuris infections have the strongest associations with anemia risk . Some concern has been raised about folic acid supplementation or fortification in high-malaria-burden settings because of a reduction in the therapeutic efficacy of sulfadoxine-pyrimethamine drugs . WhereasThe strengths of these studies include their randomized designs and high adherence to the LNS supplementation recommendations. The factorial design enabled us to examine the potential for additive effects of combined N and WSH interventions. The outcome measures were objective and therefore not subject to reporting errors or bias. All primary analyses were prespecified and data analysis was replicated by 2 investigators working with blinded data sets. The studies are limited, however, in a number of important ways. In both trials, we assessed anemia at only 1 time point. Further, the estimated prevalences of iron deficiency and iron deficiency anemia should be viewed with caution because the biomarkers of ferritin and sTfR are known to be affected by inflammation, malaria, and the presence of hemoglobinopathies , 41. We We conclude that a nutrition intervention providing LNSs with behavior change counseling focused on improving IYCF practices reduced the risk of anemia, iron deficiency, and low vitamin B-12 status in young children. The consistency in findings across both trials suggests that these results can be generalized to other populations in which anemia, iron, and vitamin B-12 deficiencies are common. Impacts on other micronutrient deficiencies were variable between sites, likely due to the timing of sample collection.nqy239_Supplemental_FileClick here for additional data file.


Alcohol and illicit drug use has been recognized as a growing problem among adolescents in Botswana. Little is known about factors affecting alcohol and drug use among Botswana’s secondary school students. To aid the design and implementation of effective public health interventions, we sought to determine the prevalence of alcohol and drug use in secondary school students in urban and peri-urban areas of Botswana, and to evaluate risk and protective factors for substance use.We performed a 72-item cross-sectional survey of students in 17 public secondary schools in Gaborone, Lobatse, Molepolole and Mochudi, Botswana. The World Health Organization’s (WHO) Alcohol Use Disorder Identification Test (AUDIT) was used to define hazardous drinking behavior. Using Jessor’s Problem Behavior Theory (PBT) as our conceptual framework, we culturally-adapted items from previously validated tools to measure risk and protective factors for alcohol and drug use. Between-group differences of risk and protective factors were compared using univariate binomial and multinomial-ordinal logit analysis. Relative risks of alcohol and drug use by demographic, high risks and low protections were calculated. Multivariate ordinal-multinomial cumulative logit analysis, multivariate nominal-multinomial logit analysis, and binominal logit analysis were used to build models illustrating the relationship between risk and protective factors and student alcohol and illicit drug use. Clustered data was adjusted for in all analyses using Generalized Estimating Equations (GEE) methods.Of the 1936 students surveyed, 816 (42.1%) reported alcohol use, and 434 (22.4%) met criteria for hazardous alcohol use. Illicit drug use was reported by 324 students (16.7%), with motokwane (marijuana) being the most commonly used drug. Risk factors more strongly associated with alcohol and drug use were reported alcohol availability, individual and social vulnerability factors, and poor peer modeling. Individual and social controls protections appear to mitigate risk of student alcohol and drug use.Alcohol and illicit drug use is prevalent among secondary school students in Botswana. Our data suggest that interventions that reduce the availability of alcohol and drugs and that build greater support networks for adolescents may be most helpful in decreasing alcohol and drug use among secondary school students in Botswana.The online version of this article (10.1186/s12889-018-6263-2) contains supplementary material, which is available to authorized users. Recent studies have shown that alcohol and illicit drug use is prevalent among secondary school students in Botswana. In 2005, the WHO-developed Global School-based Health Survey (GSHS) showed that 20.9% of surveyed 13- to 15-year-old secondary school students in Botswana “drank so much alcohol that they were really drunk one or more times,” and 7.5% used illicit drugs one or more times during their lifetime [Alcohol use at an early age is a concern due to its negative effects on the health, wellbeing, and development of young people. For example, students in several lower- to middle-income countries who have engaged in alcohol use are at higher risk for psychological distress . AlcoholFactors affecting alcohol and drug use among adolescents in different contexts are numerous and complex. In this study, we set forth to examine risk and protective factors for alcohol and drug use among secondary school students in Botswana using Jessor’s Problem Behavior Theory (PBT) as a conceptual framework. Jessor’s PBT is a cross-culturally validated model that explains problem behaviors among adolescents, including alcohol and drug use . PBT wasUnderstanding the common risk and protective factors associated with alcohol and drug use among secondary school students is imperative to the design and implementation of effective public health interventions. By furthering understanding of these issues, we hope to inform and promote the design of effective student alcohol and drug use prevention programs in this and similar settings.A 72-item cross-sectional survey was administered to secondary school students at 17 secondary schools in Botswana’s capital city, Gaborone, and surrounding villages Lobatse, Molepolole, and Mochudi. We included all seven public senior secondary schools in our target area, and randomly selected ten of the 29 public junior secondary schools in blocks to include six schools in Gaborone, two in Molepolole and one each in of the smaller communities of Lobatse and Mochudi.All students at these schools have study hall periods during which they can complete work for other classes. The Botswana Ministry of Education recommended that these periods be used for study data collection. At each junior secondary school, one study hall classroom per grade level  was randomly selected to participate in the survey. At each senior secondary school, two Form 4 study hall classrooms and one Form 5 study hall classroom were randomly selected.Surveys were administered from January 2013 to March 2013. English is the formal language of instruction in public secondary schools in Botswana. However, some students are more comfortable with the local native language, Setswana. Students were informed of the study in both Setswana and English, and their assent was demonstrated through voluntary participation in the survey. Answer sheets were labeled with unique study identification (ID) numbers instead of student names to ensure the confidentiality of student responses, and students were reassured that school officials, teachers, and parents would not have access to their survey responses. Study ID numbers were linked to names on a separate protected list, to allow for Institutional Review Board-approved follow-up interviews and focus group discussions based on responses. Teachers and school administrators were not present in the classroom while the surveys were completed. The surveys were administered during one hour-long study hall period by trained research assistants from University of Botswana, and were available in both English and Setswana.The written survey consisted of 72-items including: 1) demographic characteristics (3 items), 2) alcohol use (10 items), and 3) drug use (4 items), risk and protective factors for alcohol and drug use (43 items), and 5) motivations for alcohol use (12 items).Alcohol use was measured with the Alcohol Use and Dependency Inventory Tool (AUDIT) . AUDIT iDrug use was measured through survey items from the Botswana version of WHO’s Global Student Health Survey (GSHS). Items measured frequency of drug use during students’ lifetime, past year and past 30 days, as well as the type of drug used most often. We defined drug use in our survey as use of a drug such as marijuana, mandrax, cocaine, ecstasy, glue, benzene, methanol, prescription drugs not prescribed by a doctor, or other. Student drug use was defined as any admission of illicit drug use.Risk Factors for alcohol and drug use were measured in three domains: Models Risk , Opportunity Risk , and Vulnerability Risks . Models Risk and the Vulnerability Risks of low self-perception and low expectations for the future were measured using survey items developed and used to test the validity of the PBT model in Nairobi, Kenya . We measThe survey measured protective factors in three domains: Control Protection , Support Protection  and Models Protection (positive peer modeling). The majority of survey items were adopted from the survey tool developed and used to test the validity of Jessor’s PBT model in Nairobi, Kenya . An itemOrdinal protective and risk factor survey items were standardized using z-scores , in line with other studies that have evaluated adolescent problem behavior using the PBT framework , 20. ComComposite measures were computed for each domain. Because we do not consider the individual risk factors to be causally related to each other and do not believe that if one event occurred, the other will, we computed a composite measured by summing the risk factors or protective factors within each domain. Specifically, the domain composite measure is the average of the standardized risk factors included in each respective domain. Dichotomous risk and protective factor items were not included in the composite and subcomponent measures and were analyzed as separate risk/protective factors within their domain. Based on frequent student requests for clarification of survey question number 7, we dropped the question about parental scolding/reprimanding from the composite family monitoring variable and composite social controls protection variables.Composite and subcomponent risk and protective factor scores were also dichotomized to define high/low risk factor and high/low protective factor groups. “High risk” was defined as equal or greater than one standard deviation above the mean and “low protection” was defined by one or more standard deviation below the mean. These dichotomized scores were then used to calculate relative risk ratios to determine how substance use differed between students with higher risks and lower protections.Double data entry from paper surveys into a REDCap database with automated matching to identify errors in data entry, ensured transcription accuracy . Data weBecause the sampling design involved clustered data, students may not represent independent observations. Using a general linear nested model, we computed an intracluster correlation coefficient (ICC) to measure of the relatedness or similarity of our clustered data by comparing between cluster variability relative to between and within cluster variability. Design Effect and Effective sampling size were computed using ICC values.To examine the relationship between demographic variables and our two primary outcomes, 1) alcohol use by AUDIT score category  and 2) drug use  we computed summary statistics and conducted univariate binomial and multinomial-ordinal logit analyses. We used the Generalized Estimating Equations (GEE) method, specifying an independent correlation structure, to adjust for student-school clusters and obtain more efficient parameter estimates and better standard errors  AUDIT score of 0  vs. AUDIT score of ≥1 , and 2) AUDIT score of 0 to 4 . We also examined the relationship between drug use and 1) alcohol use (AUDIT score > 0) and 2) hazardous alcohol use (AUDIT score ≥ 5). Relative risk and 95% confidence intervals (CI) were computed with univariate binominal logit analysis using GEE methods and specifying an independent correlation structure to reflect the clustered data pattern , we conducted 4 multivariate binominal logit analysis using GEE methods that adjust for clustered data. Model 1 examined the association between drug use and all risks factors together. Model 2 added the demographic variables to the model. Model 3 added the composite protective factors to the model; and Model 4 replaced the social domain composite protective factors with the specific social subcomponent protective measures  was 0.02, rendering a design effect of 3.26, and yielding an effective sample size of All 1936 students present in class on the day of the survey opted to participate in the study. Of these participants, 1029 (53.2%) were females and 904 (46.7%) were male, and 3 (0.1%) did not specify their gender. The mean age was 16 years with an interquartile range of 14 to 17 years. There were 1173 (60.6%) participants from urban schools (Gaborone), and 763 (39.4%) went to school in a peri-urban environment . There were 402 (20.8%) Form 1 students, 371 (19.2%) Form 2 students, 360 (18.6%) Form 3 students, 566 (29.2%) Form 4 students, and 237 (12.2%) Form 5 students included in the study. Table p ≤ 0.01), they were 1.7  times more likely to drink hazardously . Males were also 2.2  times more likely to use drugs .While some students left survey questions unanswered, unanswered questions accounted for less than 2.6% of responses per item. Of the 1936 students surveyed, 1927 (99.5%) students completed the AUDIT questions, 816 (42.1%) students reported alcohol use, and 434 (22.5%) met the threshold for hazardous alcohol use. A total of 1885 (97.4%) students answered survey questions on drug use, 324 (16.7%) reported drug use, with 155 (8.0%) students reporting motokwane (marijuana) as the drug they used most often. Relative risks for adolescent alcohol use, hazardous drinking, and drug use were calculated in regard to demographic information as well as to a number of risk and protective factors and are presented in Table p ≤ 0.001), 3.5  times more likely to hazardously drink , and 3.5  times more likely to use drugs compared to those without high opportunity risk . Adolescents at high individual vulnerability risk were 1.7  times more likely to drink alcohol , 2.4  times more likely to hazardously drink , and 3.1  times more likely to use drugs .Each risk domain  was modeled separately for relation to reported behavior (See Table p ≤ 0.001), 2.1  times more likely to hazardously drink , and 2.7  times more likely to use drugs . Adolescents with low social controls protection were 2.0  times more likely to drink alcohol , 3.1  times more likely to drink hazardously , and 4.0  times more likely to use drugs . Lastly, adolescents were 10.3  times more likely to use drugs if they use alcohol hazardously as compared to no alcohol use .With regard to protective factors, particularly notable are the effects of low individual controls protection and low social controls protection. Adolescents with low individual controls protection were 1.5  times more likely to drink alcohol , 2.3  times more likely to hazardously drink , and 2.5  times more likely to use drugs .Particularly surprising to us among the results of survey data was participant responses to whether or not they “seriously considered attempting suicide in the past year.” 381 students (19.7%) answered “yes.” Of note, 36.0% of the Form 5 female students and 31.9% of Form 4 females endorsed seriously considering suicide in the past year compared to only 17.3% of male Form 5 and 14.6% of Form 4 male students. Alcohol and drug use risks were calculated in regard to the presence of suicidal ideation and it was found that adolescents reporting suicidal ideation were 1.6  times more likely to drink alcohol , alcohol availability at home , alcohol availability in the community  and low future expectations  as predictive of hazardous alcohol use. Model 1B for hazardous use vs. lower-risk alcohol use, alcohol availability in the community  and low expectations for the future were predictive of hazardous alcohol use. In both Model 2A and 2B, which control for age, gender, and school location, the same risk and protective factors remain predictive of hazardous alcohol use. Model 3 adds protective factors including support protection, models protection, individual controls protection and social control protection. Models risk and opportunity risk remain predictive in Model 3 after adding in protective factors, however the odds ratio for alcohol availability in the community decreases to 4.19 (p ≤ 0.001). Social controls protections are significantly protective in Model 3 . Model 4 controls for demographic factors, and includes subcategories of individual vulnerability risk factors and social controls protections. In this final model, predictive factors include models risk , opportunity risks including alcohol availability at home  and alcohol availability in the community , low future expectations , suicidal ideation in the past year , family monitoring , and peer controls protection related to disapproval of substance use .Multinomial regression models, controlling for socio-demographic information and adjusted for clustering by school were created to better understand the relationship between alcohol and drug use and risk and protective factors. Model 1A for hazardous alcohol use vs. no use identified models risk , alcohol availability in the community , individual vulnerability risks including low expectations of the future , and suicidal ideation within the past 12 months  as predictive factors. When age, male gender, and school location (urban vs. peri-urban) were controlled for in Model 2, the same opportunity risks and individual vulnerability risks remained predictive with similar odds ratios. Model 3, which incorporates protective factors, includes the following predictive factors: alcohol availability in the community (OR 2.22 p ≤ 0.001), social vulnerability risk , suicidal ideation , and social controls protection . The final model shows the opportunity risk of alcohol availability in the community , social vulnerability risk , seriously considering suicide in the past year  all significantly predict student drug use. Individual controls protection , and social controls protection including peer disapproval of alcohol or illicit drug use , and school attachment  are protective against drug use in Model 4.Model 1 looking at student drug use identified opportunity risks including alcohol availability at home , more than twice the rate (18.9%) found among of upper primary and secondary students surveyed during the 2010 Botswana Ministry of Education (MOE) Youth Risk Behavior Surveillance Report, and the 20.5% of secondary students ages 13–15 who reported alcohol use in the past 30 days in the 2005 WHO Global School-based Student Health Survey [While little prior data existed to explain risk factors for substance use among youth in Botswana, studies from neighboring South Africa offer opportunities for comparison. A study conducted in rural South Africa, identified several community influences associated with adolescent use of home-brewed alcohol. Positive correlations existed between alcohol use and the number of adults an adolescent knew to “engage in antisocial behaviors,” such as drug use, as well as between alcohol use and an adolescent’s perception of his or her neighborhood being in a “derelict state and being unsafe.” On the other hand, a negative correlation existed between alcohol use and perceived community affirmation . It has Within peer-reviewed literature, our findings are consistent with several other studies demonstrating the high prevalence of substance use in Botswana and Southern Africa, including among young adults and college students –31. ThisOur data suggest a number of practical solutions to target adolescents’ high consumption of alcohol and drugs in this and similar settings. Prominent risk factors for alcohol and drug use among secondary school students include opportunity risk, models risk, and vulnerability risk. Most remarkably, adolescents with easy access to alcohol, whether at home or in their community, were 3.5 times more likely to drink hazardously or use drugs


Bisphenol A is an industrial chemical compound, pervasively polluting the environment and diet, classified as an endocrine disruptor because of its interference effects on the endocrine system. In zebrafish, BPA exposure induces follicular atresia. To acquire knowledge on this atretic effect, using a qualitative and quantitative histomorphological approach, we studied zebrafish ovarian follicular stage development in response to low BPA concentrations. Results show that BPA interferes with follicular progression by affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) increases follicular recruitment by acting on primary stage follicles, (ii) forces the follicular transition from stage III to stage IV producing enlarged stage IV follicles, and (iii) induces atresia by producing atretic follicles that are peculiarly enlarged . We suggest that BPA induces atresia by the primary effect on recruitment of stage I follicles. This forces follicular progression and produces stage IV follicles that are peculiarly enlarged that undertake the atretic development. Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. It is used to manufacture plastics, epoxy resins, hard plastic bottles, and metal-based food and beverage cans. It is a pervasive industrial chemical compound, broadly present in the environment and diet ; hence, κB), among the others deeply impairing the transcriptional activity [α, ERβ, and GPR30) and androgen receptors [BPA is one of the endocrine disruptors (EDCs) whose (anti)estrogenic and antiandrogenic activity, widely documented in the past, was exerted by nuclear and extranuclear mechanisms , 8. Thisactivity , 11. Epiactivity –16, incleceptors –20. In tin utero exposure to low doses of BPA disrupts early oogenesis in mice and in rhesus monkey [In mammals, the ovary is sensitive to disruption by EDCs, especially when exposure occurs during intrauterine –23 or eas monkey , 26, whis monkey , 28. Ints monkey . In ratss monkey  and altes monkey . In agres monkey .However, the published reports on the occurrence and/or severity of the follicular effects of BPA show contrasting data, presumably reflecting differences in the experimental approaches .D. rerio) has recently emerged as an important tool for monitoring water pollution [μg/L, similar to environmental concentration) promotes follicular atresia and anovulation. These effects were not observed at higher BPA concentrations (10 and 20 μg/L). At a molecular level, we showed that 5 μg/L BPA promotes no transcriptional effect on genes involved in steroidogenesis  and oocyte growth  but significantly downregulates the expression of genes involved in ovarian development  [lhcgr), and acquisition of maturational competence  with an increase in apoptotic gene expression (caspase3 and tumor protein 53).Since the aquatic environment is a major sink for EDCs and fishes are particularly vulnerable to exposure to EDCs, the zebrafish  , productμg/L BPA effects inducing follicular atresia, using a qualitative and quantitative histomorphological approach, we evaluated ovarian follicle stages in response to BPA exposure. In particular, using the ovaries collected and molecularly already analyzed by Carnevali and coworkers [In this context, to acquire knowledge on the 5 oworkers , we morpD. rerio, AB strain, wild-type background) have been exposed to BPA by Carnevali and coworkers  for 3 weeks. Each experimental group contained 12 fish. This number has been fixed in excess by G∗Power analysis using G∗Power 3.1.9.2 software which suggested a total of 40 animals  to have 0.82 as power calculation, with p value fixed to 0.05.Adult female zebrafish (ers (see ). As alrμg/L BPA) every three days.The BPA was diluted in absolute ethanol (EtOH), used as a vehicle, and added to water . Since the extensive dilution, the control group was reared only in fresh water without EtOH, according to the Organization for Economic Cooperation Development for chronic assays (OECD 2012). Furthermore, since pilot studies have shown that the bioavailability of BPA was reduced by 50% after 4 days, the treatment with BPA was performed by renewing the water tank with the diluted pollutant  were fixed in Bouin's solution.After 3 weeks of treatment, animals were lethally anesthetized with 500 mg/L MS-222  buffered to pH 7.4. For histological analyses, ovaries .μg/L) showed blocked ovulation by atresia. The intermediate (10 μg/L) and the highest (20 μg/L) BPA concentration did not induce significant changes with respect to the control group.In a previous work carried out in collaboration with Carnevali and coworkers, it has been reported that zebrafish exposed to the lowest BPA concentration  zebrafish were processed for histological examination as already reported [n = 10 half ovaries CTRL and 10 half ovaries BPA) were embedded in paraffin, sectioned (7 μm) with a microtome, processed for hematoxylin-eosin staining (using standard procedures), and observed at 100x final magnification  under a light microscope . Images were captured using a high-resolution digital camera , and, in combination with the direct observation at the microscope, these were used to analyze follicular stages. In particular, 3 serial sections out of every 10 sections  were used to identify and count follicles, as well as to analyze the follicular area size. In BPA-exposed ovaries, the follicles appeared fragile to sectioning; therefore, the analyses were carried out using 10 CTRL and 9 BPA-exposed ovaries.Ovaries from CTRL and BPA-exposed  accumulating within the oocytes. The nucleus appeared structurally regular and enlarged and contained multiple peripheral nucleoli.During vitellogenic stages, the follicles increased in size due to the accumulation of the yolk, and the zona radiata was clearly evident. The irregular structure of the nucleus was accompanied by invaginations of the nuclear envelope. At the appearance of yolk granules, the follicles were classified as early vitellogenic stage III . The follicles were classified as late vitellogenic and maturational competent stage IV when yolk granules became abundant and started to merge and the peripheral migration of the germinal vesicle was observed. In this stage, germinal vesicle breakdown occurs .Mature or spawning stage V was identified by the absence of the germinal vesicle .As previously reported , the ideFor each ovary, a minimum of 4 random sections  were used to identify and count follicles .Follicular stage I (primary growth), stage II , stage III (early vitellogenic), stage IV , and stage V (mature) were classified and counted in both experimental groups. Using the morphological mark of atresia, we also counted the atretic follicles (hence forwardly referred to as atretic).The number of follicles in each stage relative to the total follicles  has been analyzed and graphed as mean values of 3 serial sections ± SEM.The number of stage III and stage IV relative to the total vitellogenic follicles  has been analyzed and graphed. In particular, total vitellogenic follicles have been counted and classified in early (stage III) and late (stage IV) vitellogenic stages. The percentage of follicles at stages III and IV  has been graphed as mean values of 3 serial sections ± SEM.2) and graphed as mean values of 3 serial sections ± SEM.The follicular size of vitellogenic, mature, and atretic follicles from control and BPA-exposed zebrafish was analyzed by evaluating the follicular area using ImageJ software. For each ovary, a minimum of 4 random sections were evaluated and the mean area for each follicle was evaluated on 3 serial sections. Results have been expressed in square pixels (p2) from control ovaries was used as reference values. In both experimental groups, the atretic follicles with lower and upper reference area value were graphically plotted and statistically analyzed by Fisher's chi-squared (χ2) test. The larger atretic follicles  have been referred to as big atretic follicles.The mean area of atretic follicles .Fisher's μg/L BPA) promoted follicular atresia interfering with the expression of genes involved in the development and maturation of the zebrafish ovary. Moving from these results and using the same ovaries collected and molecularly analyzed in the previous study [In collaboration with Carnevali and coworkers, we have already reported that the exposure to low-dose BPA  were processed for H&E staining and used to characterize, either morphologically or morphometrically, the area of atretic follicles in response to BPA.Ovarian sections from control and 5 p < 0.01) increased the mean area of atretic follicles (3.27 ± 0.08 p2) compared to that of the control group (1.9 ± 0.08 p2) (2) as the reference value and counted atretic follicles with a higher reference area value  (2) were present in the control group. On the contrary, a significant increase in the number of big atretic follicles  was present in BPA-exposed animals. Statistical analysis (χ2 = 0.00003) demonstrated a significant higher incidence of “big atretic follicles” in BPA-exposed ovaries . The results suggest that BPA induced a follicular atresia characterized by “big-sized” follicles.We observed that BPA modified the morphology and area of the atretic follicles. Most of these follicles appeared enlarged and fragile to sectioning and contained empty areas  peripherally localized or randomly spread . The mor0.08 p2) . Specifi0.08 p2) , suggestllicles) . Only a In order to study the follicular swelling characterizing the BPA-induced atresia (BPA-atresia), we attempt to identify the follicular stage primarily targeted by the exposure. In particular, we studied the effects of BPA on previtellogenic and vitellogenic phases of follicular development. Ovarian sections from control and BPA-zebrafish were processed for H&E staining and used to analyze the morphology, number, growth, and progression of the follicular stages using a qualitative and quantitative histomorphometrical approach.μg/L) showed cortical alveolar follicles with early formation of the zona pellucida [β and environmental estrogens is the induction of vitelline envelope zp proteins [esr1 and esr2a) whereas it did not modify the steroidogenic gene transcription.In agreement with our previous study , the hisellucida . Interesproteins , 45, sugproteins  showed tp < 0.05), whereas the number of stage II follicles significantly increased (p < 0.05). Surprisingly, we observed no effect on the number of early (stage III) and late (stage IV) vitellogenic follicles, nor on the mature ones. On the contrary, the ratio between early and late vitellogenic follicles apparently decreased after BPA exposure, and the atretic follicles were numerically higher . This suggested the BPA ability to promote atresia by forcing the vitellogenic phase.Growing evidences demonstrate that BPA affects the early follicular growth and numerically increases the atretic follicles , 39, 46.To verify this hypothesis, we investigated the BPA effects on stage III-IV transition by counting the number of stage III and IV follicles relatively to total vitellogenic ones.p < 0.05) the percentage of early vitellogenic stage III vs. the control group (p < 0.05) in BPA-exposed animals  previously reported in our experimental model [esr2a was observed in ovaries of Gobiocypris rarus exposed to BPA (50 μg/L for 5 weeks) and was associated with the exclusive presence of previtellogenic and atretic follicles [esr2a/b in zebrafish led to an arrest of folliculogenesis at previtellogenic stage II [esr1 and esr2b are expressed in the zebrafish follicle, exclusively in somatic cells, the esr2a gene is responsive to BPA and is expressed in both somatic and germinal cells, and it is the most abundantly expressed form during follicle growth [pgrmc) and acquire responsiveness to LHCGR-stimulated maturation thus developing into mature follicles. The molecular data previously reported, using ovaries collected from the same animal groups analysed herein [lhcgr, pgrmc, and esr1). The data evidenced that BPA exposure promoted follicular atresia interfering with the maturational phase.Altogether, the above-reported results demonstrate that BPA exposure elicited a double effect, particularly on previtellogenic (follicular recruitment and growth) and vitellogenic phases, suggesting potential impacting effects on ovarian reserve and atresia. Indeed, BPA forced the transition from stage I to stage II as well as from stage III to stage IV  and significantly increased the number of “big atretic follicles.” The high incidence of stage II follicles suggested further interference on follicular development. We believe that the premature development of stage II follicles slowed down their vitellogenic entry by interfering with the transition from previtellogenic to vitellogenic phase. Likely, this occurred because of the transcriptional downregulation of estrogen receptors . The efficiency of such increase was significantly higher in BPA-ovaries than in control groups (p < 0.01) demonstrating that BPA promoted follicular swelling during the vitellogenic phase. However, the follicular area of stage V was comparable to that of control stage IV indicating that the maturation physiologically occurred. Indeed, no mature follicle with abnormal size was observed in control or exposed ovaries.In both experimental groups, the follicular area of stage IV  significAlthough further studies will be necessary to define if the described effects by BPA are transient or permanent, the results reported herein suggest that BPA exposure primarily affects early follicular stages. We show that BPA forces the recruitment of stage I follicles as well as the follicular transition from stage III to stage IV with the production of stage IV follicles that are abnormally enlarged and big atretic follicles. We suggest that BPA forces the early follicular recruitment and peculiarly induces atresia by pushing follicular growth mechanisms associated with the vitellogenic phase. Interestingly, it has been reported that BPA interferes with the transcriptional expression of vitellogenin and cell-to-cell junctional proteins , 50 whicThe morphological observations reported here demonstrate that BPA interferes with follicular progression affecting the previtellogenic and vitellogenic phases. In particular, BPA exposure (i) interferes with the recruitment of stage I follicles by pressing stage I to stage II transition, (ii) forces the follicular transition from stage III to stage IV, (iii) slows down follicular progression from the previtellogenic to vitellogenic phase, (iv) enlarges late vitellogenic stage IV follicles, and (v) induces atresia by producing atretic follicles that are peculiarly enlarged .We suggest that BPA promotes atresia by impairing follicular recruitment. We propose a potential model of follicle atresia peculiarly responsive to BPA  in whichOf course, further analysis needs to decode clearly the molecular mechanisms promoting atresia responsive to BPA, although our data unequivocally show that ovary zebrafish is sufficiently sensitive, to verify the effects of environmental concentration of BPA by simple histomorphological evaluation of late vitellogenic and atretic follicular size.


The stressors affecting chickens in farming systems differ from those confronting their wild ancestors; thus, the ability to cope with an outdoor environment may have been modified. There is a growing body of literature that recognises the importance of allowing chickens to express their natural behaviour by providing outdoor access. However, studies investigating the effects of offering outdoor access to housed meat chickens in the tropics are scarce. Therefore, the effects of allowing chickens outdoor access, with two indoor space allowances, on their growth, behaviour and responses to stress were assessed. Some blood parameters indicated that outdoor access reduced stress, but the behavioural effects of providing outdoor access depended on indoor stocking density. Birds at the high stocking density with outdoor access were found standing for longer periods of time and those at the high stocking density and no outdoor access were observed walking less and lying more than the other three groups. It is concluded that there were welfare benefits of outdoor access, principally in terms of increased activity, but slower growth resulted in lighter birds at the end of the study.p < 0.05). Birds given outdoor access foraged more, but only at indoor low stocking densities (p < 0.05). Outdoor access reduced heterophil/lymphocyte ratio, indicating reduced stressor response. Birds with low stocking density indoors and outdoor access appeared more responsive to stressors, with elevated corticosterone and reduced spleen and bursa weights (p < 0.05). There were welfare benefits of outdoor access, principally in terms of increased activity, which were reflected in slower growth in the birds with outdoor access.Studies investigating the welfare of commercial-line broiler chickens raised in houses with outdoor access in the tropics are scarce, and none have investigated whether responses vary according to indoor conditions. Hence, we assessed the effects of providing outdoor access at two indoor stocking densities on broiler chickens’ growth, behaviour, stress responses and immunity in a tropical region of Mexico. One hundred and sixty chickens were assigned to one of four treatments in a factorial design: with or without outdoor access and low or high stocking density indoors. Ad libitum sampling was used to build a purpose-designed ethogram. Scan sampling was used to record the number of birds engaged in each activity of this ethogram, both indoors and outdoors. Heterophil/lymphocyte (H/L) ratio and serum corticosterone levels were tested in weeks four and six of age. When the birds were 42 days old, they were slaughtered, and the bursa and spleen harvested and weighed. In an interaction between stocking density and outdoor access, birds at the high stocking density with no outdoor pens spent the least time walking and preening and more time lying ( There is growing demand for poultry meat worldwide, particularly in developing countries partly in response to population growth and growing affluence . In respChickens behavioural responses to outdoor access systems stem from their jungle fowl ancestry . These rThere is a growing body of literature that recognises the importance of allowing chickens to express their natural behaviour by providing outdoor access ,22,23,24The study was conducted at the poultry farm of the Campus de Ciencias Biológicas y Agropecuarias (CCBA-UADY), located in the central region of Yucatán, Mexico. At an altitude of 10 m.a.m.s.l., the climate is typical of the warm subhumid tropics, with rain mostly in summer, a total annual rainfall of 999 mm, and a mean annual temperature of 28 °C, potentially increasing to 40 °C in spring and summer . The winThe experiment used a two-factor (outdoor access and stocking density) factorial design. One hundred and sixty male chickens were randomly assigned to one of four treatments: low stocking density with outdoor access (LO); high stocking density with outdoor access (HO); low stocking density indoors (without outdoor access) (LI); high stocking density indoors (without outdoor access) (HI). Each treatment had four replicates (pens). Of the 40 birds in each treatment, these were allocated at random to four groups (replicates) of ten birds. Two different enclosure sizes were used to achieve the two stocking densities. This constant group size has been showed to have only small effects on behaviour in terms of observed nearest-neighbour distances and amount of space used, compared with larger group sizes . In stocThe day-old Hubbard Flex male chicks for this experiment were purchased from a commercial hatchery. All birds were initially placed inside a circular reception area with cardboard walls of 0.50 m height and wood shavings as bedding material. Bell-type drinkers and initial tray feeders were provided. Bell-type drinkers were chosen over others since they provide a higher water availability and promote natural drinking behaviour . LightinFeed and water were provided ad libitum. A three-phase feeding program was used, consisting of a starter diet from day 1–14 of age, grower diet from day 15–28, and finisher diet from day 29–42. The ingredients and nutrient composition of the diets met recommendations for Hubbard Flex chickens [2 for the high stocking density treatments, as recommended for commercial lines in hot weather conditions [2, with a treatment enclosure size of 2 m2, and the high stocking density 10 birds/m2, with a treatment enclosure size of 1 m2. Pens were constructed of galvanised chicken wire mesh to a height of 1.20 m. Four had dimensions of 1.50 × 0.67 m (high stocking density) and four had dimensions of 1.50 × 1.34 m (low stocking density), as seen in 2/pen) was provided to compensate for the area occupied by the feeder and drinker. For the outdoor access treatments LO and HO, each pen had an outdoor area of dimensions 1.5 × 5 m, which the birds could enter through a pop-hole of dimensions 0.50 high × 1.50 m wide, via a ramp of 0.50 × 1.50 m. Fences were of galvanised chicken wire mesh to a height of 1.2 m. The eight outdoor areas (5 × 1.50 m2 for each pen) were covered with natural vegetation , as seen in The poultry facility was established in two portal-framed buildings each of dimensions 6 × 6 m. The roof was of galvanised steel sheets. Pens were arranged inside to minimise position effects in both horizontal directions, although the treatments with outdoor access were necessarily on one side of the building. The stocking density of the birds at the end of the experiment was approximately 30 kg/mnditions , and hal® COOLPIX L810, Nikon Corporation, Tokyo, Japan). Notes and 88 h of digital video recordings were combined to construct an ethogram of ten mutually exclusive behaviours, as seen in Behaviour observations started when the birds were 21 days old and concluded on day 41, prior to slaughter at 42 days old. All 160 animals were observed both in the pens and outdoor areas. Prior to the main experiment, an ad libitum sampling was conducted in similar conditions during weeks 3–6 of two production cycles to determine an appropriate ethogram and recording procedure. During these preparatory observations, behaviour was continuously recorded from 06:00 to 17:00 h, one day a week , by dire® Weather Environment System, model 01057RM, Chaney Instrument Co., Lake Geneva, WI, USA) throughout the study. For outdoor access treatments, indoor temperatures were measured at different points of the pens, far from the pop-holes.During the experiment, birds in both the conventional and outdoor access groups were observed by the same researcher. Individual birds were not identified for this purpose but the numbers of birds engaged in each behaviour were manually recorded during the scan samples, performed at 10 min intervals three times a day  for one day/week when birds were 3, 4 and 5 weeks old. For outdoor access treatments, behaviour was recorded without considering if birds were inside or outside the pen. It was not possible to complete the intended observation in week 6, since some animals exceed 3 kg of body weight and started to show mild signs of leg disorders. Video recording was used to crosscheck the behaviour at 10 min intervals and increase the accuracy of the counts ,31,32. TThe day after the behavioural observations on weeks 4 and 6, eight birds per treatment (2 birds/pen) were taken to a separate room to collect 3 mL of blood from the brachial vein. Sample collection was completed within one minute postcapture to avoid handling effects on physiological parameters . Each saWhole blood was smeared on slides, which were dried, fixed and stained . Smears g. Corticosterone concentrates were measured by enzyme-linked immunosorbent assay (ELISA) using a commercial kit . All samples were run in duplicate. Absorbance was measured at 450 nm on an ELISA microplate reader , then a correction was made by manually subtracting the mean optical density of the blank wells from all readings, since the ELISA kit product manual recommended reading the optical density at 405 nm. Interassay and intra-assay coefficients of variation were 29.6% and 13.5%, respectively. The smallest detectable concentration was 17.6 pg/mL.Sera for corticosterone analysis were obtained by centrifugation for 15 min at 1000× Chickens were individually weighed before slaughter. The spleen and bursa of Fabricius of eight animals randomly selected per treatment were removed and weighed. The relative weight (RW) of the organs to body weight was determined as a ratio .2 or high: 10 animals/m2) combinations, with repeated measures  as subplots. Behaviour was analysed as the percentage of time performing a given activity in each pen, with seven observations at three times of the day for each recording day. As no significant differences were detected between the three times of the day, data were pooled, providing one value per behaviour per pen. Values for dustbathing were mathematically manipulated by taking square roots of the values, so that the residuals most closely approximated a normal distribution. Mock fighting and grooming were rarely observed, and it was not possible to transform to achieve normality or heteroscedasticity of residual plots. Descriptive data only is reported for mock fighting. For grooming, a Fisher’s exact test was used to measure differences between systems and densities. H/L ratio and serum corticosterone levels also breached the assumption of normal distribution of residuals, so a log10 transformation was applied. Data were analysed by fitting a mixed-effects model of Minitab 17 , with pen as the random term and week as the repeated measure. Performance records were analysed considering week of age separately. The assumption of ANOVA that residuals are normally distributed was tested using the Anderson–Darling test. Least square means were calculated by using the EMMEANS subcommand of SPSS Statistics 20 . A Tukey post-hoc test was used to discriminate significant differences between pairs of means when significant differences were detected overall. A p-value of ≤0.05 was considered significant in all analyses.The pen was the experimental unit for all analyses. A split-split-plot design was used to test the effects of treatments and week of age on behaviour, H/L ratio, serum corticosterone levels, bird weight and relative weight of lymphoid organs. The main plot corresponded to housing system (outdoor access or indoors) and stocking densities  and relative humidities (%) recorded during the behavioural observations are presented in The effects of system, stocking density and week of age on the behaviour of the broilers are shown in p = 0.06). If there was an outdoor area, the high indoor density increased standing, compared with the low density.Significant interactions between system and stocking density were observed, as presented in p = 0.01) in week 5 than week 4, and foraged less  in week 4, as seen in p = 0.04).A significant interaction between system and week of age was evident for foraging, as seen in p = 0.007). Mock fighting was observed only in the LO (2.38% of 210 min of observations), LO (0.95%) and LI (1.90%) treatments in weeks 3, 4 and 5, respectively.Grooming was observed more frequently in the HO treatment  than the HI treatment  in week 4 (p = 0.01), as seen in p = 0.02), as seen in Serum corticosterone levels were increased in week 6 compared with week 4 of age, as seen in p = 0.01), as seen in p < 0.001) and at high density , as seen in p = 0.001), as seen in A significant interaction demonstrated that the H/L ratio was particularly reduced by outdoor access in birds at low density on week 6 .Spleen weight and spleen relative weight were greater in chickens raised indoors, as seen in p = < 0.001), 5  and 6 , compared with those with outdoor access, and those kept at low density were heavier on weeks 4  and 5 , compared to those housed at high density, as seen in Chickens raised indoors were heavier on week 3 (1.09 ± 0.06 vs. 1.05 ± 0.06; Since outdoor access systems have not been commonly implemented in tropical areas of Latin America, the development of a catalogue of behaviours to assess the welfare of chickens was an initial objective of this project.Locomotion was much reduced in the HI treatment, with there being no difference between the other three treatments. Locomotion plays an important role in leg health, which is facilitated by both walking and running . PhysicaThe greatest number of chickens were found lying in the HI treatment. Although lying is good for welfare if birds are comfortable, with healthy broilers spending up to 76% of their time lying , severalAmong the comfort behaviours, preening was observed more frequently in the low than high stocking density when no outdoor access was provided, although stocking density had no effect if outdoor access was available. Birds usually prefer to preen themselves indoors ,50 and iOutdoor access increased foraging, as expected, but only at the low indoor stocking density, demonstrating a reluctance of birds at the high density to forage. Foraging, as the appetitive phase of feeding , comprisThe H/L ratio tends to be reduced in subtropical birds in the winter season , due to Serum corticosterone levels were increased in week 6 of age when outdoor access was provided, and birds were kept at low density. Concentrations of corticosterone may increase in response to induced physical activity  or bioloSpleen and bursa weights and spleen relative weight all tended to be greater in chickens raised indoors than those raised with access to an outdoor area. Decreased weight of lymphoid organs in poultry has been associated with high levels of stress-induced serum corticosterone. Chronically elevated corticosterone concentrations elicit involution of lymphoid organs such as spleen and bursa of Fabricius by the depletion effect on lymphocytes from germinal cells with dysregulated immune responses, related to immunosuppression ,17,70. AChickens raised indoors and at low density had higher body weight. In contrast, birds with outdoor access were more active with slower growth as a result. This effect could be explained by the greater frequency of feeding at low density and less locomotion when no outdoor access was provided, which was also reduced with increasing density. The density results in weeks 4 and 5 are in accordance with a previous study, which reported that body weight of broilers reared in cages with different stocking densities was increased as stocking density decreased . ClearlyThis study assessed the effects of housing system and stocking density on behaviour and stress indicators of commercial-line broilers in a tropical region. The main effect of outdoor access on behaviour was to increase locomotion, which was greatly restricted when birds were kept at high stocking densities indoors, causing them to stand less, preen less and lie down more. Birds given outdoor access foraged more, but only if kept at low indoor stocking densities. Birds offered outdoor access showed evidence of reduced stress, although those kept at low stocking density inside and given outdoor access showed some evidence of fear at the end of the experiment. Birds with outdoor access generally gained less weight than those kept permanently indoors, reflecting increased activity.