Dosage‐related beneficial and deleterious effects of ginsenoside Rb1 on mouse oocyte maturation and fertilization and fetal development

Ginsenoside Rb1 (GRb1), the major saponin component of ginseng root, has a wide range of therapeutic applications for various diseases. Previously, our group showed that GRb1 triggers ROS‐mediated apoptotic cascades in mouse blastocysts, leading to decreased cell viability and impairment of pre‐ and postimplantation embryonic development, both in vitro and in vivo. In this study, we further found that GRb1 exerted dose‐dependent effects on oocyte maturation and sequent development in vitro. Oocytes preincubated with 25 μg/mL GRB1 displayed significantly enhanced maturation and in vitro fertilization (IVF) rates, along with progression of subsequent embryonic development. In contrast, treatment with 50 and 100 μg/mL GRB1 led to impairment of mouse oocyte maturation, decreased IVF rates, and injurious effects on subsequent embryonic development. In vivo, intravenous injection of 1 mg/kg body weight GRb1 significantly promoted mouse oocyte maturation, IVF, and early‐stage embryo development after fertilization while administration of 5 mg/kg body weight GRb1 led to a marked decrease in oocyte maturation and IVF rates concomitant with impairment of early embryonic development in our animal model. In terms of the mechanisms underlying the regulatory effects of GRb1 demonstrated increased intracellular reactive oxygen species (ROS) production and apoptosis in the 100 μg/mL GRb1 treatment group. However, we observed a significant decrease in total intracellular ROS content and inhibition of apoptosis events in the 25 μg/mL GRb1 treatment group, signifying that the intracellular ROS content serves as a key upstream regulator of GRb1 that influences its dose‐dependent beneficial or deleterious effects on oocyte maturation and sequent embryonic development. For further clarification of the mechanisms underlying GRb1‐triggered injurious effects, oocytes were pretreated with Ac‐DEVD‐CHO, a caspase‐3‐specific inhibitor, which effectively blocked injury to oocyte maturation, fertilization, and sequent development. In sum, study findings highlight the potential involvement of p53‐, p21‐, and caspase‐3‐dependent regulatory signaling cascades in GRb1‐mediated apoptotic processes.     

Hazardous effects of sanguinarine on maturation of mouse oocytes,fertilization, and fetal development through apoptotic processes

Previously, we reported that sanguinarine, a phytoalexin with antimicrobial, anti‐oxidant, anti‐inflammatory and pro‐apoptotic effects, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, causing decreased embryonic development and cell viability. In the current study, we investigated the deleterious effects of sanguinarine on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, sanguinarine significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with sanguinarine during in vitro maturation induced an increase in postimplantation embryo resorption and a decrease in mouse fetal weight. In an in vivo animal model, 1 to 5 μM sanguinarine, provided in drinking water, caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked sanguinarine‐triggered deleterious effects, clearly implying that embryonic injury induced by sanguinarine is mediated by a caspase‐dependent apoptotic mechanism. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 946–955, 2015.     

Effect of perfluorohexane sulfonate on pig oocyte maturation,gap-junctional intercellular communication,mitochondrial membrane potential and DNA damage in cumulus cells in vitro

Perfluorohexane sulfonate (PFHxS) is one of the most abundant perfluorinated compounds in the environment. Exposure to this compound has been correlated to a decrease in human fertility, although the molecular and cellular mechanisms underlying this correlation have not been described. The adverse reproductive effects of PFHxS could be based on alterations in oocyte maturation, the process rendering oocytes competent for fertilization. The aim of this study was to evaluate the effect of PFHxS on porcine oocyte viability and maturation in vitro, as well as on gap-junctional intercellular communication (GJIC) in cumulus–oocyte complexes (COCs), oocyte mitochondrial membrane potential (mΔΨ) and DNA damage in cumulus cells, as possible mechanisms of action. PFHxS caused cytotoxicity (medium lethal concentration, LC₅₀ = 329.1 μM) and inhibition of oocyte maturation (medium inhibitory concentration, MIC₅₀ = 91.68 μM). GJIC was not affected in exposed COCs. However, the mitochondrial membrane potential was significantly decreased in PFHxS-exposed oocytes at the germinal vesicle breakdown (GVBD) stage. In addition, exposure to PFHxS induced DNA damage in cumulus cells. Thus, inhibition of oocyte maturation by PFHxS could be attributed to a decreased oocyte mΔΨ at the GVBD and to DNA damage of the cumulus cells that support the oocyte.     

Effects of ochratoxin a on mouse oocyte maturation and fertilization,and apoptosis during fetal development

We previously reported that ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, and is a risk factor for abnormal embryonic development. More specifically, OTA triggers apoptotic processes in the inner cell mass of mouse blastocysts, decreasing cell viability and embryonic development. In the current study, we investigated the deleterious effects of OTA on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent pre‐ and postimplantation development both in vitro and in vivo. Notably, OTA significantly impaired mouse oocyte maturation, decreased IVF rates, and inhibited subsequent embryonic development in vitro. Preincubation of oocytes with OTA during in vitro maturation increased postimplantation embryonic resorption and decreased mouse fetal weight. In an in vivo animal model, provision of 1–10 μM OTA in the drinking water or intravenous injection of 1 or 2 mg/kg body weight of OTA decreased oocyte maturation and IVF, and had deleterious effects on early embryonic development. Importantly, preincubation of oocytes with a caspase‐3‐specific inhibitor effectively blocked these OTA‐triggered deleterious effects, suggesting that the embryonic injury induced by OTA is mediated via a caspase‐dependent apoptotic mechanism. Furthermore, OTA upregulated the levels of p53 and p21 in blastocyst cells derived from OTA‐pretreated oocytes, indicating that such cells undergo apoptosis via p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms. This could have deleterious effects on embryonic implantation and fetal survival rates, as seen in our animal models. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 724–735, 2016.     

Apoptotic effects of dillapiole on maturation of mouse oocytes,fertilization and fetal development

Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10?μM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspase-dependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.     

Inhibition of cell growth and induction of inflammation by endosulfan in HUVEC‐C cells

Endosulfan is one of the organochlorine pesticides. It has been associated with a wide range of adverse health effects. However, it is unknown whether endosulfan causes endothelial dysfunction. In the present study, we investigated the effects of endosulfan on human vascular endothelial cells. We exposed human umbilical vein endothelial cells (HUVEC‐C) to varying concentrations of endosulfan for 48 h. The results showed that endosulfan lowered cell viability and inhibited cell proliferation in a dose‐dependent manner. Flow cytometric analysis showed that endosulfan at 60 μM induced G1 cell cycle arrest, a response attributed to down‐regulation of CDK6 and pRb dephosphorylation. We observed that endosulfan at 40 and 60 μM induced a considerable percentage of cells to undergo apoptosis, as detected by Annexin‐V binding assays. Endosulfan reduced mitochondrial transmembrane potential, leading to the release of cytochrome c into the cytoplasm; meanwhile, endosulfan also inhibited the mRNA expression level of survivin, which resulted in the activation of caspase‐3. These results indicated that the intrinsic mitochondria‐mediated pathway was involved in apoptotic process. Exposure to endosulfan increased the secretion and mRNA expression levels of inflammation factors interleukin (IL)?6 and IL‐8, suggesting that endosulfan could cause inflammation. Overall, these findings suggested that endosulfan is toxic to HUVEC‐C cells, resulting in endothelial dysfunction. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1785–1795, 2016.     

三氯化铬对小鼠卵母细胞成熟和体外受精的影响

采用小鼠卵母细胞体外培养 ,体外受精的方法研究了三氯化铬对小鼠卵母细胞成熟和受精能力的影响 .结果表明 ,三氯化铬可以抑制卵母细胞第一极体的释放 ,降低小鼠超排卵数和卵母细胞的存活率和体外受精率 .对小鼠体内生发泡破裂没有影响 ,但可以抑制体外培养卵母细胞的生发泡破裂 ;随着在正常培养液中培养时间的延长 ,卵母细胞的第一极体的释放率和体外受精率 (除了 6.0 mg· kg-1组外 )均有显著提高 ,且与对照组相比已经无显著性差异 .结果提示 ,三氯化铬可以破坏卵母细胞的成熟 ,降低卵母细胞的受精能力 ,具有明显的生殖毒性     

Apoptotic effects on maturation of mouse oocytes,fertilization and fetal development by puerarin

Previously we identified puerarin, an isoflavone compound, as a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to retardation of embryonic development and cell viability. In the current study, we investigated whether puerarin exerts deleterious effects on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, puerarin caused significant impairment of these processes in vitro. Pre-incubation of oocytes with puerarin during in vitro maturation led to increased post-implantation embryo resorption and decreased mouse fetal weight. In an in vivo animal model, intravenous injection with or without puerarin (1, 3 and 5?mg/kg body weight/day) for 4 days caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked puerarin-triggered deleterious effects, clearly implying that embryonic injury induced by puerarin is mediated by a caspase-dependent apoptotic mechanism. These results clearly demonstrate that puerarin has deleterious effects on mouse oocyte maturation, fertilization and subsequent embryonic development in vitro and in vivo.     

Differential effects of herbicides atrazine and fenoxaprop-ethyl,and insecticides diazinon and malathion,on viability and maturation of porcine oocytes in vitro

Exposure to pesticides may be a major cause of reproductive dysfunction in humans and animals. Atrazine and fenoxaprop-ethyl, widely used herbicides, and malathion and diazinon, organophosphate insecticides, are considered only slightly toxic to vertebrates; however, there is evidence of greater effects on reproductive function. The aim of this study was to evaluate the effect of these pesticides on oocyte viability and in vitro maturation. Gametes were matured in increasing concentrations of the pesticides and then stained with MTT to evaluate viability and bisbenzimide to assess the maturation stage, in the same oocyte. Atrazine had no effect on viability but maturation was significantly reduced, while fenoxaprop-ethyl affected both parameters. The insecticides affected viability and maturation but to a different degree. The four pesticides showed a more pronounced effect on maturation than on viability, due to a blockage at germinal vesicle stage.     

Effects of citrinin on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo

The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. An earlier study by our group shows that CTN has cytotoxic effects on mouse embryonic stem cells and blastocysts, and is associated with defects in their subsequent development, both in vitro and in vivo. Here, we further investigate the effects of CTN on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. CTN induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryo development. Treatment of oocytes with 5 microM CTN during in vitro maturation (IVM) led to increased resorption of postimplantation embryos, and decreased placental and fetal weight. Using an in vivo mouse model, we show that consumption of drinking water containing 5 microM CTN results in decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. To our knowledge, this is the first study investigating the impact of CTN on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Oocyte maturation, fertilization and embryonic growth in vitro

As the oocyte develops into an embryo, cytological and metabolic events follow one another in an accurate and successive sequence. Meiosis resumes in the ovarian follicle, parallel to cytoplasmic and membrane maturation, from the onset of the ovulatory LH discharge. Only a fully mature oocyte will be recognized and penetrated by a fertilizing sperm, to ensure rapid and synchronous male and female pronuclear growth and early embryonic development. In vitro, the resumption meiosis is easily obtained once the oocyte is withdrawn from the inhibitory influence of the follicle. Cytoplasmic and membrane maturation may however be impaired, leading to fertilization failures or anomalies such as triploidy and even impaired embryo viability. Human in vitro fertilization is nowadays routinely carried out with a high success rate, but in vitro embryonic growth to the blastocyst is still unsatisfactory even with oocytes matured in the ovary, and major improvements are needed to reach optimal viability. Many studies have now been published on human oocyte maturation, fertilization and the growth of embryos in vitro. We give only a brief account of them, due to limited space, and have therefore included topics of most relevance to assisted conception as opposed to those more involved with academic research.     

Effect of organochlorine pesticides on maturation of starfish and mouse oocytes.

Methoxychlor, lindane, and dieldrin are organochlorine pesticides that have been described as altering different reproductive functions in mammals and in invertebrates. However, few data have been published concerning the effects these pesticides have on oocyte maturation and fertilization. The aim of this study was to determine whether these compounds could affect maturation of mouse and starfish oocytes. We observed that germinal vesicle breakdown (GVBD) in starfish oocytes was significantly inhibited by the pesticides. Furthermore, formation of the first meiotic spindle and extrusion of the first polar body were also altered in mouse as well as in starfish. Our results suggest that the three pesticides act on common intracellular targets in invertebrates as well as in vertebrates.     

Injury effects of ginkgolide B on maturation of mouse oocytes,fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1–6 μM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3–6 μM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.     

Zearalenone exposure affects mouse oocyte meiotic maturation and granulosa cell proliferation

Zearalenone (ZEN) is a metabolite of Fusarium and is a common contaminant of grains and foodstuffs. ZEN acts as a xenoestrogen and is considered to be cytotoxic, tissue toxic, and genotoxic, which causes abortions and stillbirths in humans and animals. Since estrogens affect oocyte maturation during meiosis, in this study we investigated the effects of ZEN on mouse oocyte meiotic maturation and granulosa cell proliferation. Our results showed that ZEN‐treated oocyte maturation rates were decreased, which might be due to the disrupted cytoskeletons: (1) ZEN treatment resulted in significantly more oocytes with abnormal spindle morphologies; (2) actin filament expression and distribution were also disrupted after ZEN treatment, which was confirmed by the aberrant distribution of actin regulatory proteins. In addition, cortical granule‐free domains (CGFDs) were disrupted after ZEN treatment, which indicated that ZEN may affect mouse oocyte fertilization capability. ZEN reduced mouse granulosa cell proliferation in a dose‐dependent manner as determined by MTT assay and TUNEL apoptosis analysis, which may be another cause for the decreased oocyte maturation. Thus, our results demonstrated that exposure to zearalenone affected oocyte meiotic maturation and granulosa cell proliferation in mouse. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1226–1233, 2015.     

Hazardous impacts of silver nanoparticles on mouse oocyte maturation and fertilization and fetal development through induction of apoptotic processes

Silver nanoparticles (AgNPs) are antibacterial materials widely used in numerous products and medical supplies. Previously, we showed that AgNPs trigger apoptotic processes in mouse blastocysts, leading to a decrease in cell viability and impairment of preimplantation and postimplantation embryonic development in vitro and in vivo. In the present study, we further investigated the hazardous effects of AgNPs on mouse oocyte maturation, in vitro fertilization (IVF), and subsequent preimplantation and postimplantation development in vitro and in vivo. Data from in vitro experiments revealed that AgNPs impair mouse oocyte maturation, decrease IVF rates, and induce injury effects on subsequent embryonic development to a significant extent. In an animal model, intravenous injection of AgNPs (5 mg/kg body weight) led to a significant decrease in mouse oocyte maturation and IVF concomitant with impairment of early embryonic development in vivo. Importantly, pretreatment with N‐acetylcysteine effectively prevented AgNP‐triggered reactive oxygen species (ROS) production and apoptosis, clearly suggesting a critical role of ROS as an upstream initiator or key regulator of AgNP‐induced hazardous effects on oocyte maturation and sequent embryonic development. Furthermore, preincubation of oocytes with Ac‐DEVD‐cho, a caspase‐3‐specific inhibitor, effectively prevented hazardous effects, highlighting the potential involvement of caspase‐dependent apoptotic signaling cascades in AgNP‐mediated events. Expression levels of p53 and p21 of blastocysts were upregulated upon preincubation of mouse oocytes with AgNPs. Our collective results imply that cell apoptosis in mouse blastocysts derived from the AgNP‐pretreated oocytes via intracellular ROS generation, which is further mediated through p53‐, p21‐, and caspase‐3‐dependent regulatory mechanisms.     

Methylglyoxal has injurious effects on maturation of mouse oocytes,fertilization, and fetal development,via apoptosis

Methylglyoxal (MG) is a metabolite of glucose. The serum MG level is increased in diabetic patients, and MG is implicated in diabetic complications related to embryonic development injury. We previously reported cytotoxic effects of MG on mouse embryonic stem cells and blastocysts, and a further association with defects in subsequent development. Here, we further investigate the effects of MG on oocyte maturation and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, MG induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with MG during in vitro maturation (IVM) led to increased resorption of post-implantation embryos and decreased fetal weight. Experiments with an in vivo mouse model disclosed that consumption of drinking water containing 10–20 μM MG led to decreased oocyte maturation and in vitro fertilization, as well as early embryonic developmental injury. Finally, pretreatment with a caspase-3-specific inhibitor effectively prevented MG-triggered injury effects, suggesting that embryo impairment by MG occurs via a caspase-dependent apoptotic process.     

Maternal benzo[a]pyrene exposure is correlated with the meiotic arrest and quality deterioration of offspring oocytes in mice

Benzo[a]pyrene (BaP) is a polycyclic aromatic hydrocarbon (PAH) in particulate matter that has a diameter of ≤2.5 μm (PM2.5). Studies have demonstrated that BaP exposure causes oocyte meiotic arrest in mice. However, whether BaP exposure also affects oocyte maturation in offspring remains unclear. To test this, female mice were administered BaP before pregnancy to generate BaP-exposed offspring. Our findings showed that BaP exposure reduced the in vitro maturation and increased the abnormalities of meiotic apparatus in offspring oocytes. In addition, BaP exposure reduced the mitochondrial content and intracellular ATP generation, induced early apoptosis, increased reactive oxidative species accumulation and the genomic DNA 5-methylcytosine (5mc) level in offspring oocytes. Along with the abovementioned defective parameters, maternal BaP exposure further compromised the embryo developmental competence of offspring oocytes. In summary, our study demonstrated that maternal BaP exposure compromised offspring oocyte maturation and quality.     

The effect of glucocorticoids on mouse oocyte in vitro maturation and subsequent fertilization and embryo development

Increased glucocorticoid levels, due to medical therapy or stress-related, may affect reproduction via the hypothalamus–pituitary-axis or directly at the oocyte level. We examined the effects of natural (corticosterone) or synthetic (dexamethasone) glucocorticoids on mouse oocyte maturation and underlying changes in extracellular signal-regulated kinase (ERK) phosphorylation patterns. Fertilization and progression up to the blastocyst stage were also evaluated. Oocytes were exposed to corticosterone or dexamethasone (0, 0.25, 2.5, 25 or 250 μM) for 17 h during in vitro maturation. After maturation, ERK-1/2 activation in oocytes was assessed by SDS–PAGE and immunoblotting, and fertilization and developmental capacity were examined in vitro. Corticosterone exposure during oocyte maturation significantly decreased progression to metaphase II, fertilization and embryo development at the highest concentration. Corticosterone caused a concentration-dependent inhibition of ERK-1/2 activation, with the highest concentration resulting in considerable inhibition of oocyte ERK-1/2 phosphorylation and no blastocyst development. In contrast, dexamethasone had no effect on maturation, fertilization and cleavage, and no effect was seen on ERK-1/2 phosphorylation. Based on these in vitro findings, high glucocorticoid levels may have consequences for subsequent development, although a short exposure to physiologic or stress-related glucocorticoid levels may not represent a hazard to meiosis progression of the oocyte.     

Chronic toxicity of commercial chlorpyrifos to earthworm Pheretima peguana

A chronic toxicity study was conducted in earthworms (Pheretima peguana) exposed to soil spiked with chlorpyrifos at concentrations of 0, 0.1, 1, 10, and 100 mg/kg soil dry matter for 7, 14, and 28 days. The integrity of the coelomocyte lysosomal membrane, nervous system, and male reproductive tissue was monitored using, respectively, the neutral‐red retention assay, acetylcholinesterase (AChE) enzyme assay, and histomorphology of spermatogenic cells in the seminal vesicles and cocoon production (at 28 days after 28 days’ exposure). Chlorpyrifos decreased the coelomocyte neutral‐red retention time (NRRT) significantly (p < 0.05) at concentrations > 0.1 mg/kg soil as early as day 7 of exposure and was dose‐ and time‐dependent. Chlorpyrifos inhibition of AChE activity was greater at day 7 than at day14 (p < 0.05) indicating possibly nerve recovery. Chlorpyrifos induced concentration‐dependent damage to spermatogenic cells and cytophores in premature stages. The number and size of premature, maturing, and fully mature spermatogenic stages were increased at low concentrations (<1 mg/kg) but a number of these maturation stages declined at higher concentrations (10 and100 mg/kg) on day 28. The most severe effects were observed in the maturing and fully mature stages at the highest chlorpyrifos concentration, and this had an adverse impact on cocoon production and cocoon viability. Collectively, the results suggest induction of widespread effects on multiple organ systems in P. peguana exposed to chlorpyrifos. Although NRRT and AChE activity were the most sensitive of the biomarkers, cocoon production and cocoon viability could still be considered as diagnostic tools for monitoring effects from low‐dose long‐term chlorpyrifos toxicity and for evaluating population effects. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1450–1459, 2016.