N,O-羧甲基壳聚糖对小白鼠精子畸形的影响

Effects of N,O-carboxymethyl chitosan on abnormality of spermatozoon in mice

目的:观察能明显促进禽类等动物的生长发育,改善肉质的新型饲料添加剂N,O-羧甲基壳聚糖对昆明种小白鼠精子畸形的影响,分析其应用在畜牧生产中的安全性。方法:实验于2005-06-22/2005-08-06于南京农业大学动物医学院药理组实验室完成。挑选体质量25～28g的小鼠50只,以随机数字表法分为生理盐水组、环磷酰胺组、N,O-羧甲基壳聚糖(南京龙源天然多酚合成厂提供,商品名为毕罗喜,质量浓度为105g/kg)2,4,8g/kg剂量组,每组10只。N,O-羧甲基壳聚糖2,4,8g/kg剂量组分别给予相应剂量的N,O-羧甲基壳聚糖,溶于1mL生理盐水,2,4g/kg剂量1次灌服,8g/kg剂量分2次灌服,每次间隔约3h;生理盐水组给予生理盐水1mL;环磷酰胺组给予环磷酰胺0.04g/kg,溶于1mL生理盐水。给药前禁食10h,不禁水。连续灌胃5d,给药后30d麻醉下处死,取两侧附睾按Wyrobek方法涂片,观察小白鼠精子畸形状况。评估标准参照农业部兽药评审委员会办公室编写的兽药试验技术规范汇编(2001年)。评估内容参照沈建忠主编的动物毒理学(中国农业出版社,2002年),包括各种畸形的精子形态:不定形、胖头、香蕉头、无钩、双头(多头)和双尾(多尾)等,及其分类计数。每组随机选取5只小鼠,计算总畸形率。精子畸形率(%)=畸形精子总数/检查精子总数×100。结果:最终25只小鼠进入结果分析,每组5只。①环磷酰胺组的精子畸形率远高于生理盐水组和N,O-羧甲基壳聚糖2,4,8g/kg剂量组(6.10%,2.48%,2.46%,2.64%,2.78%,P<0.01);N,O-羧甲基壳聚糖2,4,8g/kg剂量组与生理盐水组比较,差异无显著性意义(P>0.05),且各剂量组之间差异也没有显著性意义(P>0.05)。②各组精子畸形主要表现为头部或尾部异常。以无定形和胖头居多,占35.48%(292/823)和29.28%(241/823);其次是无钩形及香焦形多见,占19.68%(162/823)和13.49%(111/823);偶尔可见双头双尾形,只占2.07%。结论:N,O-羧甲基壳聚糖精子畸形试验结果呈阴性,说明其对雄性生殖细胞没有明显的遗传毒性。

AIM： To observe the effect of N, O-carboxymethyl chitosan, a kind of new highly effective green feed additive, which can obviously promote growth of animals, especially birds, and improve the quality and dainty of their muscles, on abnormality of spermatozoon in Kunming mice, and analyze the safety of its application in livestock. METHODS： The experiment was conducted in the Pharmacological Laboratory of Animal Medicine College, Nanjing Agricultural University from 22^nd June to 6^th August 2005. Fifty mice of 25-28 g body mass were randomly divided into normal saline group, cyclophosphamide group, 2, 4 and 8 g/kg N, O-carboxymethyl chitosan （provided by Nanjing Longyuan Natural Poly-phenol Synthesis Factory, the commercial name was Bi Luo Xi, the mass concentration was 105 g/kg） groups with 10 mice in each group. The N, O-carboxymethyl chitosan groups were infused with 2, 4, 8 g/kg N, O-carboxymethyl chitosan after dissolved in 1 mL normal saline, respectively; the 2, 4 g/ks groups were infused once, the 8 g/kg group were infused twice with 3 hours interval; the normal saline group was infused with 1 mL normal saline; the cyclophosphamide group was given 0.04 g/kg cyclophosphamide after dissolved in 1 mL normal saline. The mice were fasted 10 hours before administration. After 5 days administration, the mice were killed after the drugs were given 30 days, and the abnormal spermatozoon was observed by Wyrobek smear method. The evaluation standards were abided by the Veterinary Drug Test Regulation Assemble （2001） complicated by the Committee on the Assessment of Veterinary Drug, Ministry of Agriculture; the contents were accordance with A nimtd Toxicology written by Shen Jianzhong and published by China Agriculture Press in 2002 including the shapes of various abnormal spermatozoon： Amorphous, banana-shaped, hookless, bighead, double （multiple） heads and double （multiple） tails and so on. Five mice of each group were selected to calculated the total abnormality rate： abnormality rate of spermatozoon （%）=total number of abnormal spermatozoon/total number of spermatozoon examinedx100. RESULTS： There were 25 mice involving in the result analysis with 5 mice in each group.①The abnormal rate of the cyclophospbamide group were significantly higher than the normal saline group and three doses N, O-carhoxymethyl chitosan groups （6.10%, 2.48%, 2.46%, 2.64%, 2.78%, P 〈 0.01）; No significant difference was found among the normal saline group and three doses N, O-earboxymethyl chitosan groups （P 〉 0.05）, so was the three doses N, O-carboxymethyl chitosan groups （P〉0.05）. ②The main forms of the abnormality of spermatozoon occurred in head or tail abnormality; and amorphous and big head occupied the most 35.48% （292/823） and 29.28% （241/823）; followed by hookless and banana-shaped 19.68% （162/823） and 13.49% （111/823）; few double-head or tail were found （2.07%）. CONCLUTION： This experiment result is negative, indicating there is not obvious inherited toxicity of N,O-carboxymethyl ehitosan to male procreation cells.