【作者】 潘晓亮；

【导师】 许梓荣； 李大全；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 养羊业在新疆畜牧业中占有重要地位,绵羊数量居全国首位,新疆是我国最大的棉花生产基地,棉花产量占全国的40%,大量棉花副产品用作牛羊饲料。随着棉粕和棉籽壳大量应用,绵羊尿结石频发,公羊尿结石成为地方病,不少牧场和羊群公羊尿结石发病率达40%左右,给养羊业造成了重大经济损失。但是棉花副产品诱发公羊尿结石的研究甚少及其机理尚未阐明。本研究追踪观察饲喂棉粕和棉籽壳的生产羊群尿结石的发病情况,在此基础上,通过三个饲养试验,应用棉粕、棉籽壳诱发公羊尿结石,进而从棉粕、棉籽壳养分含量、结石的化学组成、血液和尿液相关生化指标、泌尿系统的病理变化等方面探讨了公羊尿结石形成机理。本研究的主要内容包括以下七方面：第一、追踪观察生产羊群(2538只)尿结石发病情况,并对饲养管理、病理变化、血液尿液相关生化指标测定与分析；第二、在分析追踪观察生产羊群的基础上,进行以下三个饲养诱发试验：1)选择4月龄公羊36只,随机分为3组每组12只,分别饲喂含棉粕10%、20%、30%日粮,预试期7天后,进行了为期60天的正试期；2)选择36只4月龄公羊,随机分为3组每组12只,对照组每天饲喂含7%棉粕的基础日粮,试验一组每天饲喂300g棉籽壳+基础日粮,试验二组每天饲喂450g棉籽壳+基础日粮；3)选择48只4月龄公羊,随机分为4组每组12只,对照组饲喂不含棉副产品的日粮,预试期7天后,正试期分为两阶段,第一阶段(30天)：对照组(17.5%豆粕精料),试验一组(17.5%棉粕替代豆粕的精料),试验二组(17.5%棉粕替代豆粕的精料+150g棉壳),试验三组为试验组(17.5%棉粕替代豆粕的精料+150g棉壳+相对于试验二组70%饮水)；第二阶段(90天)：对照组(按无棉粕精料饲喂),试验一组(400g棉粕+无棉粕精料),试验二组(400g棉粕+400g棉壳+无棉粕精料),试验三组(400g棉粕+400g棉壳+无棉粕精料+相对于试验二组70%饮水)；对以上三个诱发试验分别检测血液、尿液相关指标以及观察尿残渣结晶形态、数量、类型等。第三、诱发结石羊的血液尿液生化指标及尿结晶形态变化。第四、诱发结石羊的尿结石显微观察和化学组成分析。第五、诱发结石羊的泌尿系统病理变化分析。第六、以小鼠为动物模型,研究棉酚对小鼠血液指标及泌尿系统损伤的影响。第七、采用体外细胞分离培养技术,探讨棉酚对小鼠肾小管上皮细胞损伤机制。研究结果表明：1、生产公羊群在为期3个月内,每天平均采食800g棉籽壳+含12%棉粕的精料,公羊尿结石发病率高达37.2%；2、饲喂不同水平棉粕,在60天内未发现明显尿结石的病羊,但发现残渣中的尿结晶随时间和棉粕含量的增加呈增大趋势；3、饲喂不同水平棉籽壳,在60天内未发现明显尿结石的病羊,但屠宰发现尿残渣中的尿结晶随着时间的延长和棉籽壳含量的增加呈增大趋势；4、饲喂不同水平棉粕和棉籽壳,在120天饲养期内,发现试验二组尿结石发病率为25%,试验三组尿结石发病率为16.7%,但发病较早。5、尿结石中P.Mg.K含量与血清相应元素的相关性,得到结石中磷、钾、镁元素与血清相应元素相关系数分别是：Rp=0.750(中度相关)、RK=0.959(显著性相关)RMg=0.883(高度相关)。6、结石成分分析表明棉粕和棉籽壳诱发雄性细毛羊尿结石的主要成分是MgKPO4、MgKPO4·6H2O和Mg2P2O7(焦磷酸镁),首次发现绵羊肾结石中含有Mg2P2O7(焦磷酸镁)成分。7、病理切片分析表明：棉粕和棉籽壳饲喂引起绵羊肾脏和膀胱的严重损伤,泌尿系统出现炎症病理变化,为尿结石形成创造有利条件；8、组织切片结果表明：棉酚对小白鼠的肾脏有不同程度的毒害作用,可造成肾脏不同程度的肾间质出血、肾小管上皮细胞梗死、颗粒变性坏死,棉酚会引起小鼠肾脏组织有一定程度的损害作用；9、通过分离培养小鼠肾小管上皮细胞,研究棉酚对肾小管上皮细胞的影响,结果表明棉酚对肾小管上皮细胞凋亡率与棉酚浓度和作用时间呈现正相关；随着棉酚作用时间的延长乳酸脱氢酶(LDH)的泄漏量增大；肾小管上皮细胞Na+K+-ATP酶(NKA)、Ca2+Mg2+-ATP酶(cMA)活力随棉酚浓度的升高而降低。本研究结果提示：棉粕和棉籽壳引起的雄性细毛羊尿结石是一种慢性中毒性的矿物质营养代谢疾病,本试验充分证明了在产棉区雄性细毛羊尿结石的形成主要是由棉粕和棉籽壳引起,棉粕和棉籽壳诱发尿结石的主要因素是棉酚和磷、钾、镁过高。通过结石成分与血清相应元素相关性分析,说明血清中磷、钾、镁过高与结石形成有密切关系。棉酚造成肾脏肾小管和肾小球的损害,血液中磷、钾、镁过高引起尿液中代谢性的矿物质离子趋向饱和,使结晶增大、泌尿系统梗阻,最终出现尿结石。更多还原

【Abstract】 Sheep husbandry occupies an important position in Xinjiang stockbreeding industry. Xinjiang is China’s largest production base of cotton and a large number of cotton by-products used as cattle feed. With a large number of applications of cottonseed meal and cottonseed hull, urolithiasis of male wool sheep becomes endemic, which resulted in significant economic losses. But little is known about the mechanism.The study observed the prevalence situation of urolithiasis by feeding cottonseed meal and cottonseed hull. Furthermore, through three feeding trials, the formation mechanism of sheep urinary calculi was discussed by application of cottonseed meal, cottonseed hull to induce urolithiasis analysis of nutrient content of the cottonseed meal and cottonseed hull, chemical composition of stones, blood and urine biochemical parameters, pathological changes of the urinary system.The main contents include the following seven aspects:first, with the observation of incidence of urolithiasis, we analyzed and the feeding management, pathology, and biochemical parameters; Second, on the basis of the observation, three feeding induced test was designed:1) 36 male fine-wool sheep (4 months old), randomly divided into 3 groups, were fed with cottonseed meal 10%,20% 30% of the diet, respectively. After pre-trial period of 7 days, the trial was conducted 60-day; 2) 36 male fine-wool sheep (4 months old) were randomly divided into 3 groups. The control group was fed daily with basal diet (7% cottonseed meal), test group 1 fed with the basal diet+300g cottonseed hull, test group 2 fed the basal diet+450g cottonseed hull; 3) 48 male fine-wool sheep (4 months old) were randomly divided into 4 groups. The control group was fed the diet without cotton by-products. After pre-trial period of 7 days, the trial period were divided into two phases. The first phase (30 days):Group 1 was the control group (17.5% soybean meal in concentrate), test group 1 (17.5% cottonseed meal in concentrate), test group 2 (17.5% cottonseed meal concentrate+150 g cottonseed hull), test group 3 for the experimental group (17.5% cottonseed meal in concentrate+150 g cottonseed hull+ drinking 70% water of test group 2); the second stage (90 days):the control group (the concentrate without cottonseed meal), test group 1 (400g cottonseed meal+the concentrate without cottonseed meal), test group 2 (400g cottonseed meal+400 g cottonseed hull+the concentrate without cottonseed meal), test group 3 (400g cottonseed meal+400 g cottonseed hull+the concentrate without cottonseed meal+drinking 70% water of test group 2). In above trials, blood, urine and urinary-related indicators were detected and urinary crystal morphology, number and types were observed. Third, biochemical changes of blood and urine in sheep with urolithiasis were detected and crystal morphology of stone was observed. Fourth, urolith was observed by microscope and chemical composition analyzed. Fifth, pathological changes of the urinary system in sheep with urolithiasis were analyzed. Sixth, using mice as animal model, we studied the effect of gossypol on blood parameters and the urinary system injury. Seventh, using cell culture in vitro, the injury mechanism of gossypol on murine renal tubular epithelial cell was discussed.The results show that:1. When male sheep were fed daily containing 800g cottonseed hull+the concentrate containing 12% cottonseed meal in a period of 3 months, the incidence of urolithiasis was up to 37.2%;2. No sheep with urolithiasis was found during 60 days when fed with different levels of cottonseed meal, but urinary crystallization had a increase trend with increase of time and cottonseed meal;3. No sheep with urolithiasis was found during 60 days when fed with different levels of cottonseed hull, but crystallization in urine had a increase tendency with increase of time and cottonseed hull levels;4. During 120-day feeding period, the incidence of urolithiasis in test group 2 was 25% while test group 3 was 16.7% when fed with different levels of cottonseed meal and cottonseed hull.5. In urinary calculi, the contents of phosphorus, potassium, magnesium have the relationship with the corresponding elements in serum. The correlation coefficient were:RP= 0.750 (moderate correlation), RK=0.959 (significant corelated), RMg=0.883 (highly correlated)6. Urolith composition analysis showed that the main component included MgKPO4, MgKPO4·6H2O and Mg2P2O7 (magnesium pyrophosphate). It was firstly found there was Mg2P2O7 in sheep kidney calculi.7. Pathological analysis showed that the kidneys and bladders were seriously damaged and the pathological changes of inflammation was observed in urinary system in sheep with urolithiasis induced fed with cottonseed meal and cottonseed hull;8. Histological results showed that gossypol had toxicity on the kidney of mice, which can cause various degrees of renal interstitial hemorrhage, infarction of renal tubular epithelial cells, granular degeneration and necrosis. So gossypol could cause certain extent of damage on mouse kidney tissue.9. By isolating renal tubular epithelial cells in mice to study the effect of gossypol on the renal tubular epithelial cells, the results indicated that the renal tubular epithelial cell apoptosis induced by gossypol was positively correlated with gossypol concentration and reaction time. With the extension duration of treatment of gossypol, the rate of lactate dehydrogenase (LDH) leakage increased, Na+-K+-ATP enzyme (NKA), and Ca2+-Mg2+-ATP enzyme (CMA) activity was decreased with the gossypol concentration increased.The results suggested that urinary stone caused by high dose of cottonseed meal and cottonseed hull in male fine-wool sheep is a kind of a chronic toxicity of mineral nutrition in metabolic diseases. This study proved that urolithiasis of male fine-wool sheep was mainly caused by feeding with cottonseed meal and cottonseed hull. The main factors of urinary stones were high phosphorus, potassium and magnesium. The analysis of the relationship with the elements of stone composition and the mineral elements of serum indicated that high phosphorus, potassium and magnesium in serum were closely related to urolith formation. Gossypol could cause the damage of kidney tubular and glomerular, the saturation of mineral ions in urine and crystallization, the obstruction of urinary tract and finally information of urinary stones as serum contained high phosphorus, potassium and magnesium.更多还原