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玉米霉菌毒素
分为两类：
A类：田间毒素（赤霉烯酮、呕吐毒素、T-2毒素）；
B类：仓储毒素（黄曲霉毒素、赭曲霉毒素）。
注：1、赤霉烯酮：玉米赤霉烯酮具有雌激素作用，主要作用于生殖系统，妊娠期的动物(包括人)食用含玉米赤霉烯酮的食物可引起流产、死胎和畸胎。
食用含赤霉病麦面粉制作的各种面食也可引起中枢神经系统的中毒症状，如恶心、发冷、头痛、神智抑郁和共济失调等。
2、呕吐毒素：人畜摄入了被呕吐毒素污染的食物/饲料后，会导致厌食、呕吐、腹泻、发烧、站立不稳、反应迟钝等急性中毒症状，严重时损害造血系统造成死亡，但不同的动物对呕吐毒素的敏感程度不一，猪是最敏感的动物。
研究表明，呕吐毒素可能对免疫系统有影响，有明显胚胎毒性和一定致畸作用。
3、T-2毒素：T-2毒素能刺激皮肤和粘膜，引起口腔与肠道粘膜溃疡与坏死，导致呕吐和腹泻;毒素进入血液中能产生细胞毒作用，损伤
血管内皮细胞，破坏血管壁的完整性，使血管扩张、充血、通透性增高，引起全身各器官出血。
4、黄曲霉毒素：黄曲霉毒素可引起肝细胞变性、坏死、出血;影响DNA、RNA的合成与复制;抑制细胞分裂、蛋白质、脂肪的合成与线粒体的代谢;破坏溶酶体的结构和功能;还具有致癌、致突变和致畸形性等。
5、赭曲霉毒素：急性中毒反应为精神沉郁，食欲减退，体重下降，肛温升高。
消化功能紊乱，肠炎可视黏膜出血，甚至腹泻，脱水多尿，伴随蛋白尿和糖尿。
妊娠母畜子宫黏膜出血，往往发生流产。
6、伏马毒素：
伏马毒素可以引起人畜急性中毒和慢性毒性，并具有种属特异性和器官特异性，不同的动物产生的疾病不同，相应的耐受限量也有很大差别。
玉米的霉菌毒素污染一、总论玉米里的霉菌毒素是感染玉米果穗的特定霉菌所产生的。
霉菌可能存在却不产生霉菌毒素，但如果没有霉菌存在，霉菌毒素是不会产生的。
养猪生产者所关心的霉菌毒素包括黄曲霉毒素、脱氧雪腐镰孢菌烯醇（又称呕吐毒素或DON）、串珠镰孢菌素（伏马菌素）、玉米赤霉烯酮（F2毒素，镰孢菌毒素），有时候还包括赭曲霉毒素和T-2毒素（镰孢毒素）。
许多穗腐病都会滋生能够产生毒素的霉菌。
包括镰孢菌穗腐病、赤霉穗腐病以及黑曲霉穗腐病。
依阿华地区二孢菌穗腐病比较常见，但目前为止，尚未发现这种病会产生霉菌毒素。
各种穗腐病所适应的环境条件不同，因此，不同的年份发生的穗腐病类型也可能会不同。
尽管天气条件是影响霉菌感染的重要因素，但生产者却可以采取一些措施来控制感染发生的程度。
例如，玉米害虫控制可以防止害虫破坏子实，并预防因此造成的霉菌感染。
对玉米进行筛选，清除破碎或损坏的子实，并且保证相对湿度低于14%，这样可降低霉菌滋生风险。
新谷进仓之前先清仓，确保存储环境清洁，这样也可避免霉菌经陈玉米传染给新玉米。
黄曲霉毒素可能算是最受关注的霉菌毒素了，因为它对人类和动物都构成健康威胁。
由于黄曲霉毒素可在肉品当中积累，因此这是一个不容忽视的公共健康问题。
为了杜绝猪肉产品出现黄曲霉问题，商品猪饲料当中的黄曲霉毒素含量不得超过20ppb（十亿分之一）。
呕吐毒素，或称DON（脱氧雪腐镰孢菌烯醇），在饲料中含量1至3ppm时，会降低采食量和增重速度，年轻猪只最易受影响。
牛和家禽对呕吐毒素的耐受力远高于猪。
DON常常与另外一种霉菌毒素，玉米赤霉烯酮，同时出现，因为这两种毒素是同一种霉菌产生的。
伏马毒素是一个家族，包括多种霉菌毒素，马和驴对这种毒素最敏感。
对于猪，建议饲料中伏马毒素总含量要低于10ppm。
赭曲霉毒素主要影响肾脏，但同时也会对其它组织和器官造成损伤。
建议猪饲料中含量低于0.2ppm。
T-2毒素不象DON那么常见，但毒性更强，会造成拒食、呕吐以及肠道损伤。
生猪玉米赤霉烯酮中毒的诊治汇报人：日期:•疾病概述•临床症状与病理变化•治疗措施目录•预防与控制策略•案例分析01疾病概述0102玉米赤霉烯酮的危害玉米赤霉烯酮对动物具有生殖毒性、免疫抑制和胃肠道毒性，可导致生殖器官异常、繁殖性能下降、生长迟缓和胃肠道疾病。
玉米赤霉烯酮是一种真菌毒素，由某些特定的霉菌在玉米或其他谷物上生长时产生。
食欲不振、体重下降、呕吐、腹泻、皮肤炎症、生殖器官异常（如阴道和子宫颈水肿、睾丸萎缩等）。
中毒症状生猪摄入了含有过量玉米赤霉烯酮的饲料或水源，导致机体摄入过量毒素而中毒。
原因中毒的症状及原因疾病的分布与流行玉米赤霉烯酮中毒在全球范围内均有发生，特别是在潮湿、温暖、玉米种植和生猪养殖密集的地区。
流行该病的发生与气候条件、土壤类型、作物收割和贮存方式以及饲料加工方法等因素有关。
流行季节主要在作物收割和贮存期间，这时玉米赤霉烯酮污染最为严重。
02临床症状与病理变化主要症状包括发情不规则、假孕、不孕、流产、死胎、子宫炎等生殖系统问题，以及食欲不振、体重下降、皮毛粗糙等消化和皮肤问题。
主要症状包括性欲减退、精子质量下降、睾丸萎缩等生殖系统问题，以及食欲不振、体重下降、皮毛粗糙等消化和皮肤问题。
临床症状公猪母猪母猪生殖系统出现炎症和子宫糜烂，卵巢和子宫的浆膜出现出血点和淤血，子宫颈和阴道黏膜出现充血和水肿。
公猪生殖系统出现炎症和睾丸萎缩，睾丸和附睾出现萎缩和变性，前列腺和精囊腺出现萎缩和变性。
病理变化根据病猪的临床症状和病理变化，结合饲料成分分析，可以初步诊断为玉米赤霉烯酮中毒。
诊断需要与其它生殖系统疾病进行鉴别，如子宫炎、阴道炎等。
同时还需要与其它消化系统疾病进行鉴别，如胃肠炎、肝炎等。
鉴别诊断诊断与鉴别诊断03治疗措施一旦发现生猪出现玉米赤霉烯酮中毒的症状，应立即采取药物治疗，以减轻病情和缩短病程。
早期治疗对症治疗联合用药根据病情轻重和个体差异，采用不同的药物和剂量进行治疗，以达到最佳疗效。
霉变玉米的危害霉变玉米有何危害：霉菌生长需要适合的温度、湿度、氧气及能源。
当湿度大于85%,温度高于25时，霉菌就会大量迅速生长，并产生毒素。
霉变玉米产生的毒素主要有：黄曲霉菌、赤霉烯酮、伏马霉素、T-2毒素、赭曲霉毒素及呕吐霉素等。
a、黄曲霉毒素的危害—肝脏1．采食量降低或拒食。
2．生长迟滞，饲料报酬变差。
3．免疫功能降低。
4．造成肠道及肾脏出血。
5．肝胆肿大、受损和癌变。
6．影响生殖系统，胚胎坏死，胎儿畸形，盆血。
7．母猪泌乳量下降。
乳汁中因含有黄曲霉毒素，从而对哺乳小猪产生影响。
b、玉米赤霉烯酮（F-2毒素）的危害—子宫、生殖道玉米赤霉烯酮是一种具有雌激素类物质活性的毒素，主要危害种用畜禽，其中青年母猪对之最为敏感。
1.后备母猪阴部红肿，假发情。
2.母猪和后备母猪不发情。
3.保育及育肥猪增重减缓，仔猪脱肛，八字腿。
4.母猪偶发性不孕。
5.窝数少，新生仔猪体型小；新生小母猪阴部红肿。
6.假怀孕，乳腺变大，乳汁渗出，出现临产前的征兆。
7.持续性不孕，乘架其它母猪，卵巢萎缩变小。
c、呕吐毒素的危害：1.摄食量减少，增重减缓。
2.由于拒食导致饲料消耗量和增重急剧减少。
3.导致呕吐。
4.皮下及肌肉出血，降低繁殖率及抑制各种动物的免疫力。
d、赭曲霉毒素的危害—肾脏1.造成动物免疫系统功能抑制。
2.造成哺乳动物的肝脏及肾脏受损，尤其对猪和人危害更大。
3.造成母猪流产和产仔重偏轻。
4.高毒性，可致癌。
e、伏马毒素的危害—肺部、脑部1.对多数动物：降低免疫功能，损害肝脏和肾脏，降低增重，增加死亡率。
2.对猪的危害：不孕、肺水肿、呼吸困难、死亡。
3.对家禽的危害：降低食欲，免疫力下降。
4.对马的危害：贫血、脑坏死。
f、T-2毒素的危害—上皮细胞1.为毒性高的免疫抑制物质，破坏淋巴系统。
2.危害生殖系统，可引起种猪不孕、流产或产下虚弱仔猪。
3.采食量降低、呕吐、下血痢甚至死亡。
4.目前被认为是对家禽最毒的毒素，可引起口腔和肠道出血、溃疡，降低免疫力，产蛋量下降，体重减轻。
玉米赤霉烯酮对猪怀孕前后的影响一、玉米赤霉烯酮（Zearalenone）现况玉米是养猪业中使用最大宗原料之一，在收割、运送中保温不当、种植期间、久旱不雨或阴雨连绵期间，这样的环境提供玉米中霉菌滋长最佳时机。
玉米赤霉烯酮（Zearalenone）是一种引起假发情现象之霉菌毒素，由镰刀霉菌属（Fusarium）产生，在玉米种植期间就会感染并生成毒素，与新月毒素T2、呕吐毒素DON、伏马镰刀毒素FUM一样都是玉米收成前就已经存在。
其分子构造类似动情激素。
镰刀霉菌生长之条件和气候、湿度有关。
镰刀霉菌生长最好之条件为玉米含17％之水分以及存放在12～24℃下。
根据亚洲谷物中心表示：2007年至2009年收集来自中国、日本、马来西亚、菲律宾、南韩、台湾、泰国以及越南等国饲料，玉米赤霉烯酮（Zearalenone）检出率约35％，平均浓度为292 ppb，最高浓度为25 ppm。
在玉米、玉米酒粕、干草与青贮料中玉米赤霉烯酮（Zearalenone）的检出率约67％～100％。
完全混合日粮将近50％检出率，平均浓度为100 ppb；另外亚洲谷物中心也针对中国地区谷物饲料中玉米赤霉烯酮（Zearalenone）污染情形进行调查，在227件样品数中，发现含量0.1～0.2ppm占15％，高于0.2ppm则占30％。
这样高浓度加上长期喂饲下足够在猪体内引起雌激素的作用，显示在亚洲地区对玉米赤霉烯酮的控制是非常必须的。
二、玉米赤霉烯酮（Zearalenone）对母猪繁殖性能的影响：玉米赤霉烯酮（Zearalenone）为类动情激素，直接影响繁殖系统，而在饲料换肉率方面只有些许的负面影响以及生长速率变缓，但并不会常出现死亡和淘汰之情形。
当猪只食入受污染的饲料，玉米赤霉烯酮经由肠道吸收进入全身血液循环系统中。
而这些类动情素足以伤害母猪繁殖器官、抑制公猪繁殖性能。
在日粮中玉米赤霉烯酮浓度低于1ppm时，会引起类似动情激素的作用及雌性化现象。
The effect of experimental exposure to low doses of zearalenone on uterine histology and morphometry in prepubertal bitchesEwa Stopa a ,Magdalena Gaje ˛cka b ,*,Izabella Babi nska c ,q ukasz Zielonka b ,Maciej Gaje ˛cki baVeterinary Clinic,Ewa Stopa,DVM,Da ˛browskiego,I ława,Polandb Department of Veterinary Prevention and Feed Hygiene,Faculty of Veterinary Medicine,University of Warmia and Mazury in Olsztyn,Oczapowskiego,Olsztyn,Poland cDepartment of Pathophysiology,Forensic Veterinary Medicine and Administration,Faculty of Veterinary Medicine,University of Warmia and Mazury in Olsztyn,Oczapowskiego,Olsztyn,Polanda r t i c l e i n f oArticle history:Received 8January 2014Received in revised form 1April 2014Accepted 6May 2014Keywords:Zearalenone HistologyMorphometry UterusPrepubertal bitchesa b s t r a c tThe experiment involved 30clinically healthy prepubertal bitches aged approximately 70days with an estimated initial body weight (BW)of 8kg.The animals were randomly divided into two experimental groups (EI and EII)and a control group of 10animals each.Group EI was administered 50m g zearalenone (ZEN)/kg BW per os for 42days,group EII received 75m g zearalenone/kg BW per os for 42days,and the control group was admin-istered placebo per os for 42days.The bitches were hysterectomized at the end of treat-ment,and samples of uterine tissue were collected for histological and morphometric analyses.The results of the study indicate that exposure to very low doses of ZEN (100%and 150%of the NOAEL)causes simple glandular hyperplasia of the endometrium accompanied by adenogenesis,angiogenesis,and vasodilatation with the related conse-quences.The noted changes were more pronounced in group EI and less visible in group EII in comparison with group C,which could be indicative of a hormetic dose response.Ó2014Elsevier Inc.All rights reserved.1.IntroductionMycotoxin-producing molds of the genus Fusarium are the most prevalent mold fungi in all climate zones.Several Fusarium species produce toxic substances,including ZEN,a nonsteroidal estrogenic mycotoxin and a speci fic hor-mone regulating sexual reproduction of Fusarium species (sexual stage,Gibberella zeae )[1].Mycotoxicoses have been long studied in humans and animals,and they pose a serious health concern.Zear-alenone (ZEN)mycotoxicosis in farm and companion ani-mals is a speci fic problem.Dogs are among the mostpopular species of companion animals.Bitches are seasonally monoestrous animals [2]that are relatively often affected by reproductive tract disorders,such as prolonged estrus,no estrus,or ovarian cysts.It is believed that hormonal regulation of reproductive processes speci fic in bitches,which involves long progesterone and prolactin cycles and high sensitivity to endogenous and exogenous estrogens [3],plays an important role in the etiopatho-genesis of those disorders [4].The administration of hor-mones for therapeutic or biotechnological (contraception)purposes may also contribute to the development of pathological processes.The potential effects of mycotoxins (mycoestrogens)[5],which are found in pet food,on animals have not been investigated to date [6].Preliminary studies revealed high and varied levels of ZEN in many commercial pet foods,and*Corresponding author.Tel.:þ48895233773;fax:þ48895233618.E-mail address:mgaja@.pl (M.Gaje ˛cka).Contents lists available at ScienceDirectTheriogenologyjournal homepage :0093-691X/$–see front matter Ó2014Elsevier Inc.All rights reserved./10.1016/j.theriogenology.2014.05.002Theriogenology 82(2014)537–545their concentrations were quite high in some products.The presence of ZEN was determined in42out of45analyzed dog feed samples in concentrations of5.0to299.5m g/kg of the product[7].Feed contamination with the discussed mycotoxin is of particular significance for bitches that are often fed monodiets for long periods of time.ZEN is an estrogenic mycotoxin,and female dogs are particularly sensitive to estrogen.Elevated concentrations of endoge-nous and exogenous hormones also contribute to many other systemic disorders.Therefore,long-term adminis-tration of feeds containing ZEN can disrupt hormonal regulation of reproductive processes and lead to histolog-ical changes in the uterus.The uterus is particularly sensi-tive to estrogenic substances,and inflammatory processes may permanently damage this organ[8].This study was undertaken to address several problems that emerged during our previous experiments and publi-cations concerning ZEN mycotoxicosis in bitches.To the best of our knowledge,the effect of ZEA on reproductive system functions has never been studied in female dogs.The objective of this study was to determine the effect of long-term exposure to low doses(100%and150%of the NOAEL)of ZEN(subchronic mycotoxicosis)on uterine his-tology and morphometry in prepubertal bitches.2.Materials and methodsAll of the experimental procedures involving animals were carried out in compliance with Polish legal regula-tions determining the terms and methods for performing experiments on animals(opinion of the Local Ethics Com-mittee for Animal Experimentation No.37/2006issued on 24October2006).2.1.Experimental animalsThirty immature bitches aged approximately70days, with estimated average body weight(BW)of8.00Æ0.95kg, were obtained from local breeders(registered at the Polish Kennel Club)and kept in standard conditions with free ac-cess to water.Clinically healthy female dogs were divided into three groups:experimental group I(EI)(n¼10) administered ZEN per os at50m g/kg BW once daily(100%of the no-observable adverse effect level(NOAEL)[6](stimu-lating/adaptive effects)[9,10],experimental group II(EII) (n¼10)administered ZEN per os at75m g/kg BW once daily (150%of the NOAEL)(dose at which clinical symptoms are expected)[10],and control group(C)(n¼10)administered placebo without ZEN.All bitches were hysterectomized at the end of the42-day treatment period,that is,approxi-mately at the age of112days.2.2.Preparation and administration of dietsThe animals were kept in cages with ad libitum access to water and were fed standard diets tested for the presence of the following mycotoxins:aflatoxin,ochra-toxin,ZEN,a-zearalenol(a-ZEL),b-zearalenol(b-ZEL),and deoxynivalenol.Mycotoxin concentrations in the diet were evaluated with the use of common separation techniques involving immunological affinity columns and high-performance liquid chromatography(Hewlett Pack-ard,type1050and1100)[11]withfluorescent and/or UV detection techniques.Mycotoxin doses were administered daily in gelatine capsules before morning feeding for42days(subchronic ZEN mycotoxicosis).ZEN samples(Zearalenone Z-0167, Sigma Chemical Co.,Steinheim,Germany)were diluted in 300m L of96%ethyl alcohol(ethyl alcohol,96%vol.,SWW 2442–90;Polskie Odczynniki Chemiczne SA)correspond-ing to ZEN doses of50and75m g/kg BW.The resulting so-lution was introduced into the feed,placed in gelatine capsules,and stored at room temperature for12hours to evaporate the solvent.2.3.Material sampling and preparationAfter42days of oral exposure,all female dogs were anesthetized and subjected to hysterectomy.2.3.1.Material for morphometric analysisDissected uterine tissues were rinsed with cold saline solution and separated into basic anatomical parts(left and right horn of uterus and the body of uterus).All specimens were weighed and measured.The samples were prepared and processed at the Department of Veterinary Prevention and Feed Hygiene,Faculty of Veterinary Medicine,Univer-sity of Warmia and Mazury in Olsztyn,Poland.The results were presented as mean values(x)Æstandard deviation(SD).Data were processed by ANOVA. Where the null hypothesis was rejected,the differences were verified by Student’s t-test.All calculations were performed in the STATISTICA application(Statsoft).2.3.2.Material for histological analysisSamples of uterine tissue were collected for histological analysis,fixed in10%formalin,neutralized and buffered to pH 7.4,passed through a series of graded alcohols,purified in xylene,and embedded in paraffin blocks.Microtome sections were stained with hematoxylin and eosin and Periodic acid–Schiff stain according to the method proposed by McManus [12].The metrics describing the size(small,medium,and large)of a given uterine section or structure were referenced to group C values.The specimens were examined in a mini-mum of20fields of view atÂ100magnification.Microscopic images were analyzed under a100-W halogen lamp in the Olympus BX50microscope atÂ400magnification.The samples were prepared and processed at the Department of Pathophysiology,Forensic Veterinary Medicine and Admin-istration,Faculty of Veterinary Medicine,University of War-mia and Mazury in Olsztyn,Poland.3.Results3.1.Uterine morphometryStatistically significant differences(P<0.05)in the weight of the left uterine horn were noted between group C and group EII and in the weight of the right uterine horn between group C and both experimental groups(Fig.1). Highly significant differences(P<0.01)in the weight of the left uterine horn were reported between group C and groupE.Stopa et al./Theriogenology82(2014)537–545 538EI(Fig.1).The weight of the left uterine horn was higher in all experimental group animals than in control dogs.Differences at P<0.01were noted in the length of the body of uterus between group C and both experimental groups(Fig.2).No significant differences were observed in other segments of the uterus.3.2.Histological structure of the uterus in the control group 3.2.1.Region of the uterine horn proximal to the ovaries3.2.1.1.Group EI.Medium diameter(on a scale of1–6,in this case¼3)of the uterine horn.Small,medium-tall folds, mucous membrane of medium thickness.Empty,dilated lumen with forked shape.Necrotized cells and red blood cells occasionally present in the lumen.Monolayer, cuboidal superficial epithelium,locallyflat,with a ten-dency to proliferate and exfoliate.Numerous glands,mostly flat,with open ostia,with a spiral arrangement in the basal layer of the mucous membrane,tubular in the functional part.Lumen not significantly dilated.Medium-tall(on a scale of1–10,in this case¼5)glandular cells with low secretory activity,individual cells show symptoms of degeneration(Fig.3A).Mucous membrane with loose connective tissue and a predominance offibroblasts and fibrocytes in the functional part and undifferentiated cells in the basal layer.Increasing amounts of the extracellular matrix in the direction of the muscular layer.Significant focal congestion and extravasation in the lamina propria in the apical ends of mucosal folds.In the basal layer of the mucous membrane,blood vessels with spiral arrangement, dilated at the bottom of glands.Thick(approximately1/3–1/4of muscular layer thickness),homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Numerous thin-walled blood vessels surrounded by a narrow band of loose connective tissue in the vascular layer.Capillary vessels and lymphatic vessels with considerably dilated lumina.3.2.1.2.Group EII.Relatively large diameter(on a scale of 1–6,in this case¼6)of the uterine horn.Thick mucous membrane with large,smooth folds,and evident deep grooves.Empty,dilated lumen with forked shape.Sparse and shallow glands with mostly tubulararrangement, Fig.1.Uterine morphometry in prepubertal bitches(weight).*P0.05compared with control and**P0.01compared withcontrol.Fig.2.Uterine morphometry in prepubertal bitches(length).*P0.05compared with control and**P0.01compared with control.E.Stopa et al./Theriogenology82(2014)537–545539lumen insigni ficantly dilated with small mucosal content and single exfoliated cells,open ostia.Monolayer,cuboidal super ficial epithelium,locally flat.Medium-tall (on a scale of 1–10,in this case ¼5)glandular cells with low secretory activity,individual cells show symptoms of degeneration.Sparse glands irregularly distributed in the mucous mem-brane with visible nonglandular regions (Fig.3B).Glands have spiral arrangement in the basal layer and tubular arrangement in the apical ends of mucosal folds.Open ostia,very shallow glands (most reaching to the boundary of basal and functional parts of the mucous membrane).Numerous fibroblasts and fibrocytes surrounded by a small amount of the extracellular matrix in the functional part of the mucous membrane.A predominance of undifferentiated cells in the basal layer with increasing amounts of the extracellular matrix.Individual lymphocytes and white blood cells in the lamina propria.Numerous well-developed blood vessels with spiral arrangement.Medium-thick (approximately 1/3–1/4of mucous membrane thickness),homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Numerous thin-walled blood vessels surrounded by a narrow band of loose connective tissue in the vascular layer.Numerouscapillary vessels and lymphatic vessels,some with insig-ni ficantly dilated lumina.3.2.1.3.Group C.Very small diameter (on a scale of 1–6,in this case ¼1)of the uterine horn.Medium-thick mucous membrane with developed folds.Small lumen with a nearly oval shape.Small amounts of mucous discharge with single exfoliated cells of the super ficial epithelium in the lumen.Monolayer,cuboidal super ficial epithelium with single endothelial lymphocytes.Average number of very shallow glands with a spiral arrangement in the basal layer of the mucous membrane.Small lumen of the tubular part,open ostia.Medium-tall (on a scale of 1–10,in this case ¼4)glandular cells (Fig.3C),individual cells show secretory ac-tivity and symptoms of degeneration.A predominance of undifferentiated cells in the basal layer of the mucous membrane,the functional segment comprises mostly fibro-blasts and fibrocytes with small amounts of the extracellular matrix.Very strong congestion in the lamina propria,indi-vidual lymphocytes in the field of vision,infrequent white blood cells.Relatively thin (1/3of mucous membrane thick-ness),homogeneous,and clearly separated circular muscular layer with numerous acidophilic myocytes.NumeroussmallFig.3.Histopathological findings in the region of the uterine horn proximal to the ovaries in group EI dogs (hematoxylin and eosin stain).(A)Medium-tall glandular cells with low secretory activity (circle),individual cells show signs of degeneration (black arrow),Â40;group EII (B),infrequent uterine glands (forked arrows),distributed irregularly in the mucous membrane,visible nonglandular regions,Â10;group C (C),small lumina of tubular uterine glands,medium-tall glandular cells,individual cells show secretory activity (black arrow),Â40.Histopathological findings in the region of the uterine horn proximal to the body of uterus in group EI dogs (D),medium-tall glandular cells with low levels of secretory activity,individual cells show signs of degeneration (black arrow),Â60;group EII (E),medium-tall glandular cells with differentiated nuclei arranged in a single line.Low secretory activity,individual cells show signs of degeneration (black arrow),Â60;group C (F),uterine glands with tubular arrangement,shallow,with narrow lumina,open ostia (black arrowhead).Glandular cells of different height,from shallow to medium-tall,with low secretory activity (black arrow),Â40.Histopathological findings in the body of uterus in group EI dogs (G),medium-tall glandular cells with differentiated nuclei arranged in a single line along the base.Low secretory activity,individual cells show signs of degeneration (black arrow).Large number of connective tissue cells (lymphocytes (white arrow),fibroblasts (white arrowhead),and extracellular matrix (black arrowhead))and focal congestion,Â40;group EII (H),numerous lymphocytes in the lamina propria (black arrow)and strongly dilated capillary vessels,Â40;group C (I),monolayer,cuboidal super ficial epithelium.Infrequent,very shallow uterine glands.Numerous capillary vessels with spiral arrangement and signi ficantly dilated lumina (black arrow)in the apical ends of mucosal folds,Â60.E.Stopa et al./Theriogenology 82(2014)537–545540blood vessels in the vascular layer.Capillary and lymphatic vessels with dilated lumina.3.2.2.Region of the uterine horn proximal to the body of uterus3.2.2.1.Group EI.Medium diameter(on a scale of1–6,in this case¼3)of the uterine horn.Thick mucous membrane with tall folds and deep grooves.Normal,empty lumen with forked shape.Monolayer,cuboidal superficial epithelium,locallyflat.Numerous glands,mostly with spiral arrangement and tubular arrangement in the region of the ostia.Deep glands,reaching nearly to the muscular layer.Insignificantly dilated gland lumina,open ostia. Medium-tall(on a scale of1–10,in this case¼6)glandular cells with low secretory activity,individual cells show symptoms of degeneration(dark and narrow cells) (Fig.3D).Mucous membrane has loose texture with a predominance offibroblasts andfibrocytes in the func-tional part and mesenchymal cells in the basal layer.Nuclei differ in shape and size,arranged in a single line.Individual lymphocytes and white blood cells as well as histiocytes, plasma cells and macrophages in the lamina propria. Numerous,clearly visible lymphatic and capillary vessels. Thick(approximately1/3–1/4of mucous membrane thickness),homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Numerous small blood vessels(fewer than in the region of the uterine horn proximal to the ovaries) surrounded by a thin layer of loose connective tissue in the vascular layer.Capillary and lymphatic vessels with dilated lumina.3.2.2.2.Group EII.Small diameter(on a scale of1–6,in this case¼2)of the uterine horn.Thick mucous membrane with large folds and deep grooves.Empty,dilated,star-shaped lumen.Monolayer,cuboidal superficial epithelium,locally flat.Visible small foci of excessive proliferation.Infrequent glands with mostly tubular arrangement and spiral arrangement in the basal layer.Visible large nonglandular regions.Dilated lumen,open ostia,very shallow glands (along the boundary of basal and functional segments of the lamina propria).Medium-tall(on a scale of1–10,in this case¼6)glandular cells with differentiated nuclei(from dark and narrow to bright and oval),arranged in a single line (Fig.3E).Weak secretory activity,individual cells show symptoms of degeneration.Numerous connective tissue cells surrounded by small amounts of the extracellular matrix in the functional part of the lamina propria, numerous mesenchymal cells in the basal layer.Individual lymphocytes and white blood cells in thefield of vision. Sparse spiral capillary vessels and lymphatic vessels in the lamina propria.Significantly dilated capillary vessels in the lamina propria,mucous membrane congestion.Thick (approximately1/3–1/4of mucous membrane thickness), homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm. Numerous,significantly dilated blood vessels surrounded by a layer of relatively compact connective tissue in a thick vascular layer.Sparse capillary and lymphatic vessels,some with insignificantly dilated lumina.3.2.2.3.Group C.Small diameter(on a scale of1–6,in this case¼1)of the uterine horn.Medium-tall mucous membrane with large and smooth folds.Empty,narrow, star-shaped lumen.Monolayer,cuboidal superficial epithelium,locallyflat.Infrequent shallow glands with mostly tubular arrangement near ostia(Fig.3F).Narrow lumen,open ostia.Glandular cells of different height,from flat to medium-tall(on a scale of1–10,in this case¼4), with low secretory activity,individual cells show symp-toms of degeneration.The functional part of the mucous membrane has dense cellular structure with small amounts of the extracellular matrix.Numerous mesenchymal cells in the basal layer.Numerous lymphocytes and individual white blood cells in the lamina propria.Visible acute congestion.Thick(approximately1/3–1/4of mucous membrane thickness),homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Thin-walled blood vessels sur-rounded by a layer of relatively compact connective tissue in the vascular layer.Capillary vessels with dilated lumina. Numerous normal lymphatic vessels.3.2.3.Body of uterus3.2.3.1.Group rge diameter(on a scale of1–6,in this case¼6)of the body of uterus.Very thick mucous mem-brane with large,tall folds,and evident deep grooves. Narrow lumen with forked shape.Small amounts of acidophilic amorphous substance and sparse lysed eryth-rocytes in the lumen.Cylindrical to cuboidal superficial epithelium with foci of excessive proliferation and signs of exfoliation.Dilated lumen,open ostia(E.3).Infrequent,very shallow glands with spiral arrangement at the bottom, tubular in most part.Dilated lumen,open ostia.Relatively large nonglandular regions.Medium-tall(on a scale of1–10,in this case¼6)glandular cells with differentiated nuclei,from cuboidal to tall cylindrical,arranged in a single line along the base.Weak secretory activity,individual cells show symptoms of degeneration(Fig.3G).In the mucous membrane,large numbers of connective tissue cells,mostly lymphocytes andfibroblasts,with the extracellular matrix and significant foci of congestion(Fig.3G).A predominance of mesenchymal cells in the basal part.Lymphocytes and very strongly dilated capillary vessels in the lamina propria (strong congestion).Relatively thick(approximately1/3–1/ 4of mucous membrane thickness),homogeneous,and clearly separated circular muscular layer with infrequent myocytes with acidophilic cytoplasm.Thin vascular layer with loose connective tissue.Very numerous capillary vessels with somewhat dilated lumina.Strongly dilated lymphatic vessels.3.2.3.2.Group EII.Medium diameter(on a scale of1–6,in this case¼3)of the body of uterus.Thick mucous mem-brane with smooth and tall folds with deep grooves.Empty, narrow lumen with forked shape.Monolayer,cuboidal su-perficial epithelium,locally damaged,with pyknotic nuclei, degraded cytoplasm or foci of excessive proliferation. Relatively numerous glands with mostly spiral arrange-ment,very shallow.Dilated lumen,open ostia.Medium-tallE.Stopa et al./Theriogenology82(2014)537–545541(on a scale of1–10,in this case¼6)glandular cells,mostly with narrow nuclei with dense chromatin,arranged in a single line.Weak secretory activity,individual cells show symptoms of degeneration.A predominance offibrocytes andfibroblasts surrounded by a large amount of the extracellular matrix in the functional part of the lamina propria.A large number of mesenchymal cells,fibroblasts, and individualfibrocytes in the basal part of the mucous membrane.Individual lymphocytes and white blood cells in thefield of vision.Very strong congestion of the mucous membrane,in particular near the apical ends of folds.Thick (approximately1/3–1/4of mucous membrane height thickness),homogeneous,and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Vascular layer with numerous small blood vessels with dilated lumina surrounded by a band of loose connective tissue.Numerous capillary and lymphatic ves-sels,some with strongly dilated lumina(Fig.3H).3.2.3.3.Group C.Small diameter(on a scale of1–6,in this case¼1)of the body of uterus.Smooth and tall mucous membrane folds with evident deep grooves.Narrow lumen with forked shape and small amounts of acidophilic amorphous substance.Monolayer,cuboidal and cylindrical superficial epithelium(Fig.3I).A very small number of very shallow glands with a spiral arrangement near the bottom and a tubular arrangement near the ostia.Wide lumen, open rge nonglandular regions.Medium-tall(on a scale of1–10,in this case¼5)to cylindrical glandular cells with mostly narrow nuclei with dense chromatin.Nuclei positioned along the base.Weak secretory activity,indi-vidual cells show symptoms of degeneration.Numerous connective tissue cells with a predominance offibrocytes andfibroblasts in the functional part of the lamina propria and mesenchymal cells in the basal part.Individual lym-phocytes and white blood cells in thefield of vision.Very numerous spiral capillary vessels in the mucous mem-brane,with significantly dilated lumina in the apical ends of folds(congestion)(Fig.3I).Thin(approximately1/4–1/5 of mucous membrane thickness),homogeneous and clearly separated circular muscular layer with numerous myocytes with acidophilic cytoplasm.Thick vascular layer with loose connective tissue.Numerous normal capillary vessels. Lymphatic vessels with insignificantly dilated lumina.4.DiscussionOral administration of monotonic doses of ZEN to pre-pubertal bitches over a period of42days led to the permanent presence of ZEN,a-ZEL,and b-ZEL in the animals’bodies during the entire experiment(samples were collected from the same bitches)[13].The metabolism of natural estrogens, including mycoestrogens(undesirable substances),is often regarded as a detoxification process that lowers the concen-trations of the native substance(in this case,ZEN),but may also be accompanied by the production of new substances that are much more toxically active(e.g.,a-ZEL)than the native substance[14].ZEN-induced hyperestrogenism in both experimental groups led to specific morphometric changes of varied intensity.The exposure to lower ZEN doses in group EI led to a higher total increase in the weight of uterine horns than in group EII.This was probably provoked by a greater proliferative effect of a lower ZEN dose and higher nonphysiological concentrations of E2in the same animals from group EII[13].The fact that uterine horns in group EII bitches were shorter than in group EI or even group C animals indirectly attests to the above.Increasing concentrations of estrogens,including E2, stimulate the proliferation of stromal cells and the growth of glandular ducts(which accumulate glycogen)and spiral arteries.The endometrium becomes thicker,leading to the restoration of glandular epithelium that lines the uterine cavity and increased uterine weight in prepubertal bitches.In newborns and young mammals,the uterus un-dergoes vast structural and functional changes,including differentiation and development of uterine glands(ade-nogenesis).According to Cooke et al.[15],the administra-tion of progesterone(P4)or progestogens to newborn sheep and mice inhibited adenogenesis,a process that plays a key role in uterine function.This is an important observation because ZEN and its metabolites disrupt the hormonal balance,and similarly to estradiol(E2)[13],they induce conformational changes and bind to estrogen re-ceptors(ERs)[16,17].The above leads to histological changes,including in the uterus.ZEN and its metabolites are agonists for ER a(they are observed mainly in the uterus,but not in the ovaries)[17,18],and they act as partial antagonists for ER b.The ability of ZEN to bind to ERs is10-to100-fold lower than the binding ability of E2,whereas the binding ability of a-ZEL is20-fold higher than that of ZEN.According to Gaje˛cka et al.(samples collected from the same bitches)[13],ZEN,a-ZEL,and b-ZEL are potential endocrine disrupters[10]because they provoke an exces-sive release of steroid hormones(P4and E2)at different levels of development in young animals.Similar observa-tions were made by Frizell et al.[14]and Dunber et al.[19], and our results corroborate theirfindings.The exposure to low doses of ZEN(in both experimental groups)increased steroid hormone levels,in particular P4, in the initial stage of the same experiment,which was discussed in a different article[13].The exposure to E2 produced different results because elevated concentrations of E2were noted in thefinal stage of the experiment in both experimental groups in comparison with control[13]. Contrary to the hypothesis postulated by Van Cruchten et al.[20],the above led to changes in the physiological sequence of the increase in hormone concentrations during maturation(before thefirst estrus,in the middle,rather than at the end of anestrus[21]),which can produce false behavioral symptoms(false estrus),sexual maturation (specific morphotic changes in the uterus),or histological changes in estrogen-dependent tissues(e.g.,higher secre-tory activity of uterine glands)[22].Morphometric changes and proliferation of the mucous membrane lining the uterus are observed in consequence of ovarian follicle maturation,but they could also be induced by exogenous estrogens,including ZEN,that cause hyperestrogenism[13].The above increased the weight of uterine horns(Fig.1),the length of the body of uterus (Fig.2),the diameter of the uterine horn or the body of uterus,and the height and thickness of mucosal folds in both experimental groups,proportionally to the ZEN dose.E.Stopa et al./Theriogenology82(2014)537–545 542。
