动物试验,精子畸形 z-bo 2020-08-13 503

　　Sperm abnormalities in mice are genetically controlled and have high heritability. Many autosomal and X and Y sex chromosome genes directly or indirectly determine the morphology of sperm. Sperm abnormalities mainly refer to abnormal morphology. It is known that sperm abnormalities determine spermatogenesis. The result of morphological gene mutation. Therefore, morphological changes indicate changes in related genes and their protein products. The mouse sperm abnormality test can detect the impact of environmental factors on sperm production and development, which leads to a high degree of mutagenesis of known germ cells. Therefore, the test is a mutagen for human germ cell environmental factors. Can be used for detectable impact.

　　equipment and reagents

　　Unless otherwise specified, all reagents are of analytical grade and test water is distilled water.

　　1 Experimental experimental equipment.

　　2 Biological microscope.

　　3 M ethanol.

　　4 1%? 2% eosin staining solution: Weigh 1-2 g of eosin and dissolve it in 100 mL of distilled water.

　　6-8 weeks old animal (25-35g). 2 doses are divided into negative control group (solvent), positive control group and 3 test groups, with at least 5 surviving animals in each group. The substance is cyclic amine phosphate 40-60mg/(kg·d) or methyl methanesulfonate (MMS) 50mg/(kg·d), mitomycin C (MMC) 1.0-1.5mg/(kg·d) . The highest dose can be regarded as the maximum tolerated dose, or a dose group of 1/2, 1/4 and 1/8 LD50, respectively. Orally for 5 consecutive days.

　　3 operation steps

　　3.1 Animal running time: Different pathogenic variants act on different stages of sperm development, and may cause sperm deformation at different times after exposure to certain mutants. Have. Therefore, if conditions permit, the animals can be killed after 1, 4, and 10 weeks of administration of the test substance to determine the sperm morphology. Most chemical mutagens are produced in late spermatogonia or in primary spermatogonia. Because it is more sensitive to cells, it is usually done 35 days after the first dose of test substance. Take out two mice and epididymis and place them in a small beaker containing an appropriate amount of saline (1 mL) or a petri dish containing 2 mL of saline. Cut off the epididymis with ophthalmic scissors vertically with 1-2 knife, and let it stand for 3-5 minutes. Shake gently for 5 minutes. Use 4 layers of lens cleaning paper or synthetic fiber blood net bag to filter, and then suck out the filtrate smear. After air drying, fix with methanol for more than 5 minutes, and then dry. Stain with 1% to 2% eosin for 1 hour, rinse with water, and dry.

　　3.3 Microscopic examination: look for areas with low power (using green filter), clear background and little sperm overlap. Use high power to check the morphology of sperm and count the number of sperm in the complete structure. Sperm with a head but no tail (unclear outline), or if the head overlaps with other sperm or fragments, or is obviously artificial, it is not counted. At least 1,000 spermatozoa are examined for each animal.

　　Sperm abnormalities mainly appear on the head and then on the tail. The variants are divided into no hook, banana shape, thick head, amorphous, folded tail, double head, double tail, etc. Record the microscope coordinates of each sperm, record the abnormal type separately, and calculate the sperm deformation rate and the composition ratio of the sperm deformation type.

　　When distinguishing between two-headed and two-tailed variants, please pay attention to the difference between the two sperm and determine the stage of partial overlap. When it is judged to be amorphous, it is necessary to distinguish it from manual cutting and folding. Statistical methods and determination results Each dose group should be compared with the corresponding negative control group through parameter statistical methods (for example, using Wilcoxon grade). The evaluation criteria for abnormal sperm with a positive total detection method is that the abnormal rate is more than twice that of the negative control group or is statistically significant, and there is a dose-response relationship.

　　The abnormal sperm rate of the general negative control group is 0.8% to 3.4%, for your reference, but please refer to the natural deformation rate of experimental animals used in this laboratory.