α-氯代醇对雄性高原鼠兔生殖功能的影响
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摘要
若尔盖湿地是青藏高原高寒湿地生态系统的典型代表,是我国特有生物多样性关键地区之一。但近年来,若尔盖草原生态系统的结构和功能遭到破坏,其中高原鼠兔(Ochotona curzoniae)种群数量反常升高就是重要原因之一,于是高原鼠兔成了草原生态系统中的主要害兽。
本实验将1/2LD50、1/4 LD50和1/8LD50剂量的α-氯代醇(α-chlorohydrin, ACH)灌胃高原鼠兔后,采用精子计数、病理组织学观察、电镜观察、电化学发光免疫等方法研究ACH对高原鼠兔的毒性、生理生化、雄性生殖系统的结构与功能影响,以此来检测α-氯代醇对雄性高原鼠兔生殖功能的影响,以选择合适的剂量来控制高原鼠兔,保护草原生态。
用改良寇氏法测定ACH对高原鼠兔的LDso。结果表明：ACH对高原鼠兔的急性口服半数致死量为103.16mg/kg。LDso的95%可信限为85.75-124.09mg/kg。LD50的平均可信限为103.16±19.17mg/kg。根据以上结果按《中华人民共和国农药登记毒理学试验方法GB15670-1995》农药的急性经口毒性分级标准评定,ACH对高原鼠兔为中毒。
按药物对精子的致畸实验要求,将ACH根据LD5o的结果配置1/2LD50、1/4LD50和1/8LD5o浓度,经口灌胃给雄性性成熟高原鼠兔,常规饲养19天后处死,检测体重、睾丸和附睾重量、精子数量、活力和畸形率,血清中睾酮、乳酸脱氢酶-C4和总蛋白的含量,对睾丸和附睾进行了病理组织学和超微病理学观察。结果表明,ACH对高原鼠兔的体重、睾丸和附睾重量无显著影响。ACH能非常明显降低高原鼠兔精子的数量、活力和活率(P<0.01),显著增加精子畸形率(P<0.01),随着ACH剂量增加,精子无尾率会大幅增加(P<0.01)。
试验证实,ACH对高原鼠兔血清的睾酮、乳酸脱氢酶-C4和总蛋白浓度都有非常显著的影响(P<0.01)。睾酮由1.38 ng/mL增加到4.40 ng/mL,乳酸脱氢酶-C4由2006.80 U/L下降到866.20U/L,总蛋白由74.37 g/L下降到49.40 g/L。表明ACH对精子生长发育有显著影响。从病理组织学和电镜观察表明,ACH能明显减少附睾管中精子数量,生精细胞的发育停滞在精母细胞阶段,次级精母细胞数量明显增多。曲精小管壁明显增厚,精母细胞中溶酶体数量增多。
本实验通过ACH对高原鼠兔抗生育效果的结果表明,ACH能显著降低雌性高原鼠兔的活胎率,由每胎平均4.4只下降到1.05只。早期死亡胚胎数和晚期死亡胚胎数随着ACH剂量的增加而显著增加,分别由每胎平均0.15只和0.15只增加到2.55只和1.25只。本试验证实,60mg/kg体重的ACH可作为野外控制高原鼠兔种群数量的参考剂量。
Located in the eastern margin of Qinghai-Tibet Plateau, Zoige Wetland is a typical represent of alpine wetland ecosystems.But recently, the structure and function of this ecosystem is declining as results of global warming, temperature rise, plateau uplift, water level dropped and Zoige widespread over-grazing phenomenon, especially the abnormal population increasing of plateau pika (Ochotona curzoniae).It is reasonable for plateau pika to become a major vermint in grassland ecosystems, which exacerbats its degradation.
In this study,α-chlorohydrin (a-chlorohydrin, ACH) was administered to plateau pikas.The ACH effect on plateau pikas toxicity, physiology, biochemistry, male reproductive system structure and function was detected by the methods of sperm counting, HE staining, observation of lectron microscopy and electrochemiluminescence immunoassay.From the study,suitable dose of ACH will be choosed to control the amount of plateau pikas in order to protect the grassland ecosystem.
In order to meet the requirements of following-up experiments, ways to detect ACH on the plateau pika LD50 was determined according to The People's Republic of China Pesticide Registration toxicological test method (GB15670-1995). The results show the acute oral median lethal dose of ACH on plateau pika is 103.16mg/kg.While 95% confidence limit of LD50 is 75～124.09mg/kg,the average confidence interval LD50 is 103.16±19.17mg/kg. Based on the above results and the oral toxicity grading criteria, ACH was assessed to be poisoning to plateau pikas according to the acute pesticides.
Udder the experimental results of LD50, ACH was disposed to different concentrations of 1/2LD50、1/4 LD50 and 1/8LD50 then was administered to male sexual maturity plateau pikas in dose of weight by 0.2mL/10g.Pikas was sacrificed after 19 days conventional breeding.Indexes of bodyweight, testis and epididymis, sperm count, vitality and deformity rate, serum testosterone, lactate dehydrogenase-C4 and total protein content were tested. Besides, both histopathological and ultrastructural of the testis and epididymis and anti-fertility effect on plateau pika were observed.
Consequencely, ACH have very significantly reduced the number, vitality and motility of sperm in the plateau pika (P<0.01), with contrast of a significant increase in the rate of sperm deformity (P<0.01). Otherwise the sperm rumples rate has been a significant increased by the ACH increasing dose (P<0.01). However, plateau pika weight, testis and epididymis weight were not affected.
ACH have a very significant effect on the plateau pika serum testosterone, lactate dehydrogenase-C4, and total protein (P＜0.01). Index of testosterone incrased from 1.38 ng/mL to 4.40 ng/mL, while LDH-C4 decreased from 2006.80U/L to 866.20U/L and total protein decreased from 74.37g/L to 49.40g/L. This indicates ACH has a significant influence on the growth and development. It also can be indicated from the histopathological and electron microscopy that ACH can significantly reduce the sperm number in epididymal sperm tube,hold up the development of spermatogenic cells, and increase the number of secondary spermatocytes considerably. Seminiferous tubule wall became significantly thicker, and the number of cytolysosome in spermatocyte increased.
This experiment of ACH on the plateau pika anti-fertility effect confirmed that ACH can significantly reduce the female plateau pika vivi-fetus rate, from an average of 4.4 merely per fetus to 1.05. In addition,the number of early deaths and late fetal death of embryos increased significantly with increasing ACH dose,from an average of 0.15 per fetus and 0.15 to 2.55 and 1.25. This study confirmed that 60mg/kg can be a reference dose of ACH to control the amount of plateau pikas in wild.
本实验将1/2LD50、1/4 LD50和1/8LD50剂量的α-氯代醇(α-chlorohydrin, ACH)灌胃高原鼠兔后,采用精子计数、病理组织学观察、电镜观察、电化学发光免疫等方法研究ACH对高原鼠兔的毒性、生理生化、雄性生殖系统的结构与功能影响,以此来检测α-氯代醇对雄性高原鼠兔生殖功能的影响,以选择合适的剂量来控制高原鼠兔,保护草原生态。
用改良寇氏法测定ACH对高原鼠兔的LDso。结果表明：ACH对高原鼠兔的急性口服半数致死量为103.16mg/kg。LDso的95%可信限为85.75-124.09mg/kg。LD50的平均可信限为103.16±19.17mg/kg。根据以上结果按《中华人民共和国农药登记毒理学试验方法GB15670-1995》农药的急性经口毒性分级标准评定,ACH对高原鼠兔为中毒。
按药物对精子的致畸实验要求,将ACH根据LD5o的结果配置1/2LD50、1/4LD50和1/8LD5o浓度,经口灌胃给雄性性成熟高原鼠兔,常规饲养19天后处死,检测体重、睾丸和附睾重量、精子数量、活力和畸形率,血清中睾酮、乳酸脱氢酶-C4和总蛋白的含量,对睾丸和附睾进行了病理组织学和超微病理学观察。结果表明,ACH对高原鼠兔的体重、睾丸和附睾重量无显著影响。ACH能非常明显降低高原鼠兔精子的数量、活力和活率(P<0.01),显著增加精子畸形率(P<0.01),随着ACH剂量增加,精子无尾率会大幅增加(P<0.01)。
试验证实,ACH对高原鼠兔血清的睾酮、乳酸脱氢酶-C4和总蛋白浓度都有非常显著的影响(P<0.01)。睾酮由1.38 ng/mL增加到4.40 ng/mL,乳酸脱氢酶-C4由2006.80 U/L下降到866.20U/L,总蛋白由74.37 g/L下降到49.40 g/L。表明ACH对精子生长发育有显著影响。从病理组织学和电镜观察表明,ACH能明显减少附睾管中精子数量,生精细胞的发育停滞在精母细胞阶段,次级精母细胞数量明显增多。曲精小管壁明显增厚,精母细胞中溶酶体数量增多。
本实验通过ACH对高原鼠兔抗生育效果的结果表明,ACH能显著降低雌性高原鼠兔的活胎率,由每胎平均4.4只下降到1.05只。早期死亡胚胎数和晚期死亡胚胎数随着ACH剂量的增加而显著增加,分别由每胎平均0.15只和0.15只增加到2.55只和1.25只。本试验证实,60mg/kg体重的ACH可作为野外控制高原鼠兔种群数量的参考剂量。
Located in the eastern margin of Qinghai-Tibet Plateau, Zoige Wetland is a typical represent of alpine wetland ecosystems.But recently, the structure and function of this ecosystem is declining as results of global warming, temperature rise, plateau uplift, water level dropped and Zoige widespread over-grazing phenomenon, especially the abnormal population increasing of plateau pika (Ochotona curzoniae).It is reasonable for plateau pika to become a major vermint in grassland ecosystems, which exacerbats its degradation.
In this study,α-chlorohydrin (a-chlorohydrin, ACH) was administered to plateau pikas.The ACH effect on plateau pikas toxicity, physiology, biochemistry, male reproductive system structure and function was detected by the methods of sperm counting, HE staining, observation of lectron microscopy and electrochemiluminescence immunoassay.From the study,suitable dose of ACH will be choosed to control the amount of plateau pikas in order to protect the grassland ecosystem.
In order to meet the requirements of following-up experiments, ways to detect ACH on the plateau pika LD50 was determined according to The People's Republic of China Pesticide Registration toxicological test method (GB15670-1995). The results show the acute oral median lethal dose of ACH on plateau pika is 103.16mg/kg.While 95% confidence limit of LD50 is 75～124.09mg/kg,the average confidence interval LD50 is 103.16±19.17mg/kg. Based on the above results and the oral toxicity grading criteria, ACH was assessed to be poisoning to plateau pikas according to the acute pesticides.
Udder the experimental results of LD50, ACH was disposed to different concentrations of 1/2LD50、1/4 LD50 and 1/8LD50 then was administered to male sexual maturity plateau pikas in dose of weight by 0.2mL/10g.Pikas was sacrificed after 19 days conventional breeding.Indexes of bodyweight, testis and epididymis, sperm count, vitality and deformity rate, serum testosterone, lactate dehydrogenase-C4 and total protein content were tested. Besides, both histopathological and ultrastructural of the testis and epididymis and anti-fertility effect on plateau pika were observed.
Consequencely, ACH have very significantly reduced the number, vitality and motility of sperm in the plateau pika (P<0.01), with contrast of a significant increase in the rate of sperm deformity (P<0.01). Otherwise the sperm rumples rate has been a significant increased by the ACH increasing dose (P<0.01). However, plateau pika weight, testis and epididymis weight were not affected.
ACH have a very significant effect on the plateau pika serum testosterone, lactate dehydrogenase-C4, and total protein (P＜0.01). Index of testosterone incrased from 1.38 ng/mL to 4.40 ng/mL, while LDH-C4 decreased from 2006.80U/L to 866.20U/L and total protein decreased from 74.37g/L to 49.40g/L. This indicates ACH has a significant influence on the growth and development. It also can be indicated from the histopathological and electron microscopy that ACH can significantly reduce the sperm number in epididymal sperm tube,hold up the development of spermatogenic cells, and increase the number of secondary spermatocytes considerably. Seminiferous tubule wall became significantly thicker, and the number of cytolysosome in spermatocyte increased.
This experiment of ACH on the plateau pika anti-fertility effect confirmed that ACH can significantly reduce the female plateau pika vivi-fetus rate, from an average of 4.4 merely per fetus to 1.05. In addition,the number of early deaths and late fetal death of embryos increased significantly with increasing ACH dose,from an average of 0.15 per fetus and 0.15 to 2.55 and 1.25. This study confirmed that 60mg/kg can be a reference dose of ACH to control the amount of plateau pikas in wild.
引文
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