SU1329780A1 - Method of determining quality of spermatozoa - Google Patents

Description

This invention relates to animal breeding biology and is intended for laboratory and clinical research in the field of cryobiology, veterinary medicine, and medicine.

The aim of the invention is to improve the accuracy of determination.

Example. Thiazine red and ethidium bromide in an amount of 0.006 and 0.001 May / about are introduced into the nutrient medium: o. Then, sperm and nutrient medium containing fluorochrome (for example, 0.01 ml of sperm and 0.1 ml of medium) are measured in a test tube at a ratio of 1:10, carefully stirred, put a drop of the mixture on a glass slide and tightly covered with a cover slip. glass so that sperm movement is restricted. After that, the drug is placed under a fluorescent microscope (ML-2 or ML-3). In this case, excitation light with a wavelength of 365 nm is used, and f. 1- orescence observed in the region of 500-700 nm. The drug is considered under immersion with an increase of at least 90x15 in reflected light. The field of view takes into account the number (percentage) of damaged and normal sperm. At the same time, lighter spermatozoa, which are normal, are clearly visible on a dark blue background (acrosome of normal sperm is somewhat darker than the head, but lighter than the background). If the acrosome is damaged, the permeability of its membrane, thiazine red, penetrates into the acrosome, and causes its light to be damaged. yu-lettuce glow. When more than one person has acrosome damage, I congregate 0

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This violation of its morphological structure, most often the acquisition of a spherical shape, thiazin red causes its intense, bright crimson glow. When the cytoplasmic membrane of sperm cells is damaged, ethidium bromide penetrates into the cell and, by binding to DNA, causes a bright luminescence of the sperm head.

The optimum ratio in a mixture of thiazine red and ethidium bromide is determined as follows. Various samples of dyes, as well as detergent digitalanin at a final concentration of 1 mM per target, are injected into the nutrient medium (diluent of sperm of cocks LKS-1) damage to all sperm head membranes and acros

The research results are presented in the table.

For the convenience of taking into account the intensity of luminescence of damaged sperm organelles when using one or another concentration of dyes, the table shows a 5-point characteristic, where 5 points reflect the most intense luminescence of damaged acrosomes and sperm heads; 4 points - intense luminescence of one damaged organelle (for example, acrosomes) and me} its intense, but still clearly distinguishable luminescence of another (for example, head); 3 points - insufficiently intense luminescence of the head and acrosome, or sufficiently intensive luminescence of one organelle with a faint luminescence of the other; 2 points - a weak luminescence of the tin and acro-1 point - a barely perceptible luminescence of the sperm.

The data presented in the table indicate that tiazon red and ethidium bromide can be effectively used in a mixture within fairly wide limits (0.003-0.012 wt.% And 0.0005- 0.002 wt.%, Respectively). The optimal concentration of thiazin red in this mixture is 0.005-0.008% by weight, and ethidium brome is 0.0008- 0.0015% by weight. Insufficient concentration of dyes (or one of them) in the mixture, i.e. less than the lower limit, does not provide a clear differentiation of sperm structures due to weak luminescence, and an excess (above the upper limit) leads to an increase in the intensity of the background luminescence, which makes it difficult to observe. The excess of one dye (for example, thiazine red) masks the luminescence of another (ethidium bromide), introduced in insufficient concentration.

Thus, the proposed method allows, if necessary, to differentiate in the sperm preparation to normal ones (unlit dark blue head and acrosome); with a normal head (dark light, dark blue) and damaged acrosome (light salad or bright crimson luminescence); with a damaged head (a bright crimson glow) and intact acrosome (inconspicuous, dark blue); with a damaged head and a damaged acrosome (light schA with a light salad acrosome and a bright crimson head or a bright crimson glow of the acrosome and

heads); with a normal head (lighter, dark blue) and the absence of an acrosome; with a damaged head (a bright crimson glow) and the absence of an acrosome;

separately located from each other are the light of the center of sperm organelles (acrosome, head, tail).

This differentiation from terms allows you to more accurately determine and then eliminate the cause of these or other damages. This is especially important in the development of freezing and thawing of sperm, the study of the toxicity of cryoprotectants, the development of media for storing and freezing of sperm, and operating conditions.

Claims (1)

males, in clinical practice, etc. Invention Formula A method for determining the quality of sperm cells, including shifting the sperm with a nutrient medium into which ethydium bromide fluorochrome is introduced, and subsequent analysis of the stained sperm under a microscope, characterized in that, in order to improve the accuracy, fluorochrome thiazine red is added to the nutrient medium in the following ratio ingredients, wt.%: Fluorochrome thiazine red0,003-0,012 Fluorochrome ethidium bromide 0, 0005-0.002 Nutrient environment The rest of sperm quality is judged by damage to their head and acrosome.