| Di(2-ethylhexyl)phthalate(DEHP)is a plasticizer widely used in plastic products.The special molecular structure of the plasticizer DEHP makes it easy to release from plastic products,enter and pollute the air,soil and water,and eventually enter the body through dietary intake,respiration or skin absorption.The widespread use of plastic products made humans and animals widely and continuously expose to high levels of DEHP.Studies in humans have shown that DEHP exposure is associated with multiple reproductive diseases and endangers human reproduction.At present,there are few studies on the relationship between plasticizer DEHP and animal fertility,especially in vivo experiments.Therefore,it is meaningful to explore the effect of DEHP on female fertility in vivo.In addition,the overall fertility of dairy cows decreased over the last few decates,but the specific reasons still remained unclear.Furthermore,the decline tendency was more obvious in the retail dairy farms.There were many retail dairy farms in China and a variety of plastic utensils were used in the retail farming process,posing a threat to the health of dairy cows.Considering that dairy cows could contact with many plastic utensils in daily life,the risk of continuous exposure to DEHP will significantly increase in retail farm.Previous studies have shown that DEHP exposure can lead to a decline in human fertility.Therefore,this study is aimed to explore the contaction situation of plastics utensils in cow's retail farming environment,as well as the relationship between DEHP and the decline in fertility of dairy cows.In this study,we first analyzed the situation of plastic utensils exposed to the daily life of dairy cows in the farm through investigation,and tested the residual plasticizer DEHP in several common cow feeds.Next,DEHP-exposed mouse model was prepared by oral administration,then HE staining technique,oocytes in vitro maturation,oocytes in vitro fertilization,and immunofluorescence staining techniques were used to analyze the impacts of DEHP on primary follicular morphology in ovary,oocyte maturation rate,fertilization rate and oocyte oxidative stress and cytoskeleton,epigenetic modification state of oocytes,and female fertility in vivo.The main results are as follows:(1)Through field investigation and questionnaire research,it was found that plastic barrels,plastic antiskid pads,plastic basins,plastic water pipes and other plastic equipment were used during the dairy farming process both in retail farms.Moreover,the use of plastic utensils was not standardized and some plastic appliances were rarely replaced unless broken in some retail dairy farms.The residual plasticizer DEHP was detected in the cow's roughage peanut meal by liquid chromatography-mass spectrometry.Together,the results of the survey indicated that dairy cows have the risk of continuous exposure to DEHP in retail farms.(2)The mouse in vivo DEHP-exposed model was made by intragastric administration and an obvious hepatocyte edema was observed in DEHP-exposed model.The pathological changes of ovary with DEHP exposure were observed by HE staining.The results showed that DEHP exposure caused oocytes nucleus disappeared in the primary follicle,the granular cells around the oocyte were extremely loose and detached,and exhibited disorganized and disrupted morphologies.(3)The effects of DEHP in vivo exposure on oocyte maturation rate and fertilization rate in mice were analyzed by in vitro maturation and in vitro fertilization techniques.The results showed that,compared with the control group(76.75% ± 1.33%,n = 224),the maturation rate of oocytes in DEHP-exposed group was significantly reduced(50.86% ± 2.14%,n = 235);what's more,DEHP exposure also affected the in vitro fertilization rate of mouse oocytes.(64.47% ± 2.78%,n = 104 vs 83.61% ± 2.47%,n = 183).(4)The oxidative stress level,early apoptosis,cytoskeleton morphology and epigenetic modification of mouse oocytes treated with DEHP were analysed by immunofluorescence staining.The results showed that long-term continuous exposure to DEHP affected mouse oocyte cytoskeletal dynamics,induced oxidative stress,caused early apoptosis,disrupted the normal morphology of meiotic spindle,inhibited mitochondrial activity,decreased ATP content,decreased Juno expression,and caused DNA damage and altered epigenetic modification status,ultimately result in decreasing maturation and fertilization rate of mouse oocytes.In addition,the fertility of female mice significantly reduced after DEHP exposure.In summary,field investigations showed that cows exposed to a variety of plastics in their daily life in retail farm,and the detection of DEHP residue in individual feeds,the above investigations indicated that dairy cows have the risk of continuous exposure to DEHP in retail farms.Subsequent experiments in model animal mice have shown that plasticizer DEHP in vivo exposure reduces the female fertility by causing ovarian lesions,abnormal morphology of primary follicles,and damage to oocytes.This study showed that the plasticizer DEHP could reduce the female fertility,and the research results provide some guidance and reference for the establishment of standardized measures and optimization of management mode in dairy farming,as well as provide a new theoretical basis for finding ways to improve the fertility of dairy cows. |
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