The method of determining the viability of the sperm fish after cryopreservation
(57) Abstract:The invention relates to the fishing industry. The method consists in the fact that the samples of sperm before and after cryopreservation exposed to radioactive indicator of free radical reactions with subsequent measurement of the radioactivity of the samples by the method of liquid scintillation. The assessment of the damage to membranes is performed according to the level change of the content labeled free radicals, and considered viable samples, with the rate of change of the level of free radicals of no more than 30-50%. The method has sufficient sensitivity to identify, simple to execute. 2 C.p. f-crystals, 1 table. The invention relates to the fishing industry and can be used in artificial breeding of fish and other animals in the gene Bank and the securities and endangered species with the aim of preserving biodiversity.Practical use of methods of cryopreservation of sperm of animals is constrained by their imperfection and neizvjesnosti mechanisms tripolidine and ways of their correction. Modes of freezing and thawing, the increase in the concentration of intra - and extracellular salts, excessive dehydration somaticheskih membranes, from the integrity of which depends on the normal functioning of cells.To ensure high efficiency methods of cryopreservation of sperm of animals is very important with a high degree of accuracy to predict its survival after cryopreservation. It is necessary to assess the validity closewith environments and adjust their composition, sampling the most viable sperm for bookmarks for long term storage and forecasting the viability of the embryo.Currently there is not enough objective methods for determining the viability of the sperm of animals according to the degree of damage after cryopreservation.The most common way to determine the viability of the sperm after cryopreservation is to assess sperm quality of fish in its activity. The activity determined under the microscope by the ratio of the number of motile sperm with rectilinear translational motion to the total number of cells in field of view and expressed in percentage. (See Collection of scientific, technical and methodological documentation on aquaculture. M: VNIRO, 2001, S. 152-158).The method is not sufficiently reliable and reproducible due to the complexity obtained the sperm causes the death of a part of the sperm, this introduces additional error. A stable and reproducible conditions of finding sperm in the implementation of the method (temperature, pH, and others) is also difficult. In addition, sperm motility does not characterize the level of damage in the process of cryopreservation and does not always correlate with their viability and fertilizing ability, which is the main indicator of the quality of sperm.There is a method of assessing the quality of sperm, which consists in measuring the amount of heat released by the sperm at a constant temperature incubation. (See The GDR patent No. 85875, CL 45 h 29/00, 1971).However, this method is not correct, because the amount of heat released by the sperm depends on many unmanageable factors such as the degree of excitation, the maturity of the sperm, etc.Known methods of determining the quality of sperm, based on the principle of time measurement discoloration of sperm under the influence of various indicators. (See auth. mon. The USSR №375067, C And 61 D 7/02, 1973, No. 622466, And 61 D 7/02, 1978).However, these methods are unsuitable for assessing the viability of the sperm fish after cryopreservation, because salt composition closewith cf is trifaldi assessment of the level of damage to the membranes, which consists in mixing the sperm with a nutrient medium containing fluorochrome, and subsequent analysis of the stained sperm under a microscope. (See auth. mon. No. 1329780, class A 61 D 7/02, And 61 In 10/00, 1987). As fluorochrome use a mixture of two dyes - casinovegas red and ethidium bromide. In damages of the membrane of the acrosome casinoby red penetrates the acrosome and causing it to glow, and ethidium bromide, penetrating through the damaged cytoplasmic membrane of sperm, causes the illumination of his head. The quality of the sperm is estimated by the intensity of their fluorescence under fluorescent microscope according to the grading scale.However, this method is time consuming and provides only a General idea of the damaged structure membrane of sperm on a scale of not allowing a quantitative description of the level of damage.The closest way to the same destination to the claimed invention on the totality of symptoms is the way to determine the viability of the sperm, involving the assessment of the degree of damage to the membranes according to the change of cell respiration in different environments. (See auth. mon. The USSR №938990, C And 61 D 7/02, 1982). The polarographic method is the evaluation of energy systems is predelut the integrity of the outer cell membrane of the sperm by the reaction of breath antart potassium. Because intact spermatozoa amber-acidic potassium does not penetrate, increased respiration takes place only in the case of increasing the permeability of membranes.Thus, this method allows you to graphically register the measurement results and to determine the relative speed of the breath before the introduction of entrata potassium and since its introduction, the degree of damage to the membranes.For reasons that impede the achievement of specified following technical result is that the method is not available for use in the field, difficult to perform, requires expensive equipment. Furthermore, the method is indirect and is not sensitive enough, since the result of the determining to a large extent depends on the accuracy and reproducibility of the preparation of the sample.The present invention is directed to the extension and simplification of the method, reducing the complexity and improving the detection sensitivity.The technical result is achieved in that in the method of determining the viability of the sperm fish after cryopreservation, involving the assessment of the degree of damage to the membranes and determine its viability as a result of this assessment, a special indicator of free radical reactions with subsequent measurement of the radioactivity of the samples by the method of liquid scintillation, the assessment of the damage to membranes is performed according to the level change of the content labeled free radicals by the equation=(DPMop-DPMTO)100/DPMK,where is an indicator of changes in the level of labeled free radicals, %,DPMKcontent tagged free radicals in the samples of sperm prior to cryopreservation, u/min,DPMopcontent tagged free radicals in the samples of sperm after cryopreservation, u/min,and consider viable samples, with the rate of change of the level of free radicals (a) not more than 30-50%.In addition, a radioactive tracer is advisable to add to the sperm samples in the ratio of 10:1,5. Thus preferably the sperm exposed to radioactive indicator within 1-2 hoursIn the proposed method for evaluating the degree of sperm cryodamage used method of radical copolymerization. The method is focused on metabolic criterion is the intensity of free radical reactions. This method allows to measure the stress response of sperm cells, subjected to cryopreservation, by comparing the level of free radical reactions in them with the control (charania intensity redox metabolic reactions in the cells is reflected in the content of free radicals (SRS) - short-lived intermediate products of these reactions. Radicals can be considered as the driving factors of biochemical reactions. Main groups of free radicals, as defined in animal cells by various methods, characterize the two different categories of oxidative reactions: the formation of peroxy radicals in lipid membranes and the formation of intermediates redox enzymatic reactions. When the cell damage caused by various reasons, developing nonspecific "oxidative stress", including the development of chain reactions of lipid peroxidation, formation of superoxide radicals that damage the membrane structure of cells and genetic material, and at a later stage - change (usually decrease) the activity of membrane-associated enzymes and enzymes of antioxidant defense system of cells (catalase, SOD, and other). Thus, in various pathological processes induced by the reaction of a radical nature, which disrupt the steady flow of processes in the cell.The principle of the method consists in the use of water-soluble radioactive indicator monomer which, when introduced into living cells when Incubus is not removed from the cell. After fixing the membrane of sperm relative content of CF is detected according to the level of radioactivity in samples of sperm.By electron paramagnetic resonance found that CF in the sample contacted with the substance-marker. Damage detected by CF-reactions in the first 1-2 hours of exposure and then confirmed in long-term experiments to determine the viability of the sperm of other methods and their fertilizing capacity. The preferred ratio of the number of radioactive indicator to the sperm samples 10:1,5 chosen empirically.Numerous experiments revealed that the level of changes in the content of CU in the samples of sperm fish to characterize the degree of cell damage, normally does not exceed 30-35%; a level shift WED-reactions by more than 50% corresponds to the onset of pathological processes, and more significant changes lead to their death.The analysis of the level of technology has allowed to establish that not found the source, which is characterized by symptoms that are identical to all the essential features of the claimed invention. Therefore, the claimed invention meets the level of "novelty."Dopolnitelnye level of technology.Therefore, the claimed invention meets the condition of "inventive step".The proposed method is as follows.Samples of sperm before and after cryopreservation exposed to radioactive indicator of free radical reactions. Samples of sperm after cryopreservation pre-thawed in warm water. Semen add a radioactive tracer, such as14S - acrylamide activity 0.1 mccoury/ml at a ratio of 1.5:100, and incubated for 1-2 h at room temperature. Next, the samples are washed five times with ringer's solution at milliprobe filters under pressure. Then the filters with samples placed in vials for scintillation account and dried in the air during the day. Samples prepared for measurement of radioactivity by the method of liquid scintillation to determine the radioactivity. Similar operations are performed for samples of native (not frozen) sperm or native semen diluted test environment. The value of radioactivity of samples corresponds to the content of free radicalsThe assessment of the damage to membranes in the samples is performed according to the level change of the content labeled the ü changes in the level of labeled free radicals, %;DPkcontent tagged free radicals in the samples of sperm prior to cryopreservation./min;DPMopcontent tagged free radicals in the samples of sperm after cryopreservation./min.Viable believe the samples, with the rate of change of the level of free radicals (a) not more than 30-50%.The invention is illustrated by the following example.Experiments on determination of the viability of the sperm fish after cryopreservation produced with semen sturgeon, frozen in various closewith environments. Samples of sperm before and after cryopreservation were subjected to radioactive indicator of free radical reactions. Samples of sperm after cryopreservation, in vitro "Eppendorf", previously thawed by placing in a water bath with a temperature of 38-40C for 1 minute. In tubes containing 1.5 ml of sperm was added to 10 μl of radioactive indicator (14S - acrylamide activity 0.1 mccoury/ml) and kept for 1.5 h at room temperature. Next, the samples were washed five times with ringer's solution at milliprobe filters under pressure, using a water-jet pump. To do this, we selected and placed the dried on the air during the day. Before measuring the radioactivity by the method of liquid scintillation samples were prepared as follows: to the acid hydrolysis in each vial was added 0.4 ml of conc. HClO4and 0.4 ml of conc. H2ABOUT2; was placed in a thermostat (50 ° C) for 6 h; then added 2.5 ml of 1.5 M triaminobenzene to neutralize the acid and 15 ml of scintillation mixture on the basis of dioxane (solution Shaving). Determination of the radioactivity of the samples was carried out on a liquid scintillation spectrometer "Rackbeta" company LKB.The assessment of the damage to membranes in the samples was carried out according to level change of content labeled free radicals by measure and by the percentage of fertilized eggs deportirovanniy sperm and further development (before hatching). Control was native semen (without freezing). Each version of the experiment consisted of 3 replicate samples.The results are shown in the table.As can be seen from the table, the values of changes in the levels of labeled free radicals in sperm samples of more than 30-50% corresponds to a sharp decline in male fertility.Based on the above data we can conclude that the environment No. 64 has unlocks the ability deportirovanniy sperm. Createsite quality of the environment No. 63 acceptable and environments No. 56 and 52 is insufficient.Thus, the inventive method allows to determine the cryodamage in the sperm before placing samples for long term storage in the Cryobank, which contributes to reduce maintenance costs biogenesis. As a rule, cryodamage may be present in 90% of samples. On average, per year put into the Cryobank about 11000 spermatos. Securing one stated stud fees (including the cost of liquid nitrogen) is 38 rubles. Savings from prior (predzakatnogo) control tripolidine the claimed method is about 30,000 rubles.The inventive method is versatile, easy, simple to perform, has sufficient detection sensitivity and can be used to develop conditions of freezing and thawing of sperm, cryoprotectants and environments for freezing, as well as developing other ways of saving.The above data suggest the implementation of the use of the claimed invention the following cumulative conditions:- the method of determining the viability of the sperm fish after cryopreservation stated in the other animals;for the inventive method, it is described in the independent clause sets out the claims, confirmed the possibility of its implementation using the steps described in the application of tools and techniques.Therefore, the claimed invention meets the condition of "industrial applicability". 1. The method of determining the viability of the sperm fish after cryopreservation, involving the assessment of the degree of damage to the membranes and determine its viability as a result of this evaluation, characterized in that the samples of sperm before and after cryopreservation exposed to radioactive indicator of free radical reactions with subsequent measurement of the radioactivity of the samples by the method of liquid scintillation, the assessment of the damage to membranes is performed according to the level change of the content labeled free radicals by the equation=(DPOP-DTO)100/DTO,where is an indicator of changes in the level of labeled free radicals, %;DPMKcontent tagged free radicals in the samples of sperm prior to cryopreservation./min;DPMOPcontent tagged free radicals in the sperm samples after ª free radicals (a) not more than 30-50%.2. The method according to p. 1, wherein the radioactive tracer is added to the sperm samples in the ratio of 10:1,5.3. The method according to p. 1 or 2, characterized in that sperm exposed to radioactive indicator within 1-2 hours
SUBSTANCE: invention proposes applying lazin as antidote for hydrobionts in poisoning with triazoles and organophosphorus compounds. Invention can be used in piscine farms polluted with pesticides. Invention provides reducing detriment effect of pesticides for piscine farms.
EFFECT: valuable properties of antidote.