RU2352328C1 - Method for correcting vascular wall atrophy in newborn piglets suffering from anemia - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: method consists that Ferroglukin is injected intramuscularly in dosage 150 mg (2 ml) twice every 10 days. It is combined with intramuscular injections of Phosprenyl three times in different syringes into different points. For the first time Phosprenyl dosed 0.08 ml/kg is combined with Ferroglukin injected. For the second time Phosprenyl dosed 0.07 ml/kg is injected five days after the first injection of Ferroglukin. And for the third time Phosprenyl dosed 0.06 ml/kg is combined with the second injection of Ferroglukin. Additionally Hamavit is injected intramuscularly in dosage 0.015 ml/kg once a day within ten days since the first injection of Ferroglukin.

EFFECT: method allows for reduced pigs mortality, for healthier stock, for prevented weakening of animal organisms, for increased weight gain and ensured healthy breed from these animals.

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Description

The invention relates to veterinary medicine and biology, namely to angiology.

Analogues of the proposed method for the correction of the weakening of the functions of the vascular wall in newborn piglets with anemia does not exist.

In the literature there is information about the use of ferroglukin in anemia in piglets (Internal non-communicable diseases of farm animals. Edited by V.M. Danilevsky. M.: Agropromizdat, 1991. - 576 p.).

In modern veterinary medicine, phosprenyl is used as effective stimulants of the vital activity of a growing organism in phosprenyl, a drug based on polyprenol phosphates obtained from needles, and gamavit, a complex balanced means based on the placenta, containing sodium nucleate, a set of amino acids, vitamins, and salts (Deeva A.V., Salakhova ZA, Lobova TP, et al. Improving the safety and productivity of piglets when using fosprenil and gamavita. // Veterinary Medicine. - 2006. - No. 4. - p.13-15).

However, never before has a treatment complex consisting of ferroglukin, fosprenil and gamavit been used in newborn piglets with anemia in order to correct their vasopathy.

The aim of the invention is the correction of the weakening of the functions of the vascular wall in newborn piglets with anemia.

The essence of the proposed method lies in the fact that to correct the weakening of the functions of the vessel wall, newborn piglets with anemia are prescribed ferroglukin 150 mg (2 ml) intramuscularly, twice with an interval of 10 days, fosprenil intramuscularly, three times in different syringes at different points, the first time 0, 08 ml / kg simultaneously with the iron preparation, a second time at a dose of 0.07 ml / kg five days after the first injection of ferroglukin, a third time at a dose of 0.06 ml / kg simultaneously with a second injection of ferroglukin and gamavit 0.015 ml / kg intramuscularly once a day in Seven days after the first injection ferroglyukina.

The method allows to correct the weakening of the functions of the vascular wall in newborn piglets with anemia 5 days after completion of treatment. Subject to the recommendations of the proposed method, it is possible to normalize the functions of the vascular wall, which will significantly improve the number of piglets, increase weight gain, reduce mortality and subsequently receive healthy offspring from these animals.

The inventive method is as follows.

The following examination is performed to diagnose a weakening of the functions of the vessel wall of newborn piglets with anemia.

The antiaggregatory activity of the vascular wall is determined by the method (Baluda V.P., Lukyanova T.I., Baluda M.V. Method for determining the antiaggregatory activity of the wall of human blood vessels. // Labor. Delo, 1983. - No. 6. - P. 17- twenty). The basis of this method is the calculation of the degree of inhibition of platelet aggregation in plasma obtained from blood after temporary venous occlusion.

A cuff of the sphygmomanometer is applied to the vessel from which blood sampling has not yet been performed and a pressure of 10 mmHg is created. higher than systolic. After 3 minutes, 9 ml of blood is taken in 1 ml of a 3.8% sodium citrate solution. It is immediately centrifuged for 5 minutes at 1000 rpm to obtain platelet-rich plasma. The plasma is aspirated with a pipette and immediately placed in melting ice, where it is stored until the start of the study. The sample is centrifuged for 20 minutes at 3000 rpm to obtain platelet-free plasma (BTP). After counting the platelet count in platelet-rich plasma obtained before venous stasis (described in detail below), it is diluted with platelet-poor plasma (BeTP) taken before (1 test) and after (2 test) venous stasis to a concentration of 200,000 platelets / μl. The resulting mixture was shaken and incubated for 10 minutes at 20-22 ° C. Then platelets (AT) are aggregated with collagen (1: 2 dilution of the main suspension) on glass in the following manner (A. Shitikova, In: Hemostasis. Physiological mechanisms, principles for the diagnosis of the main forms of hemorrhagic diseases. Ed. By N.N. Petrishcheva, L.P. Papayan. St. Petersburg, 1999. S.49-52).

Blood is taken with sodium citrate 3.8% in a 9: 1 ratio, centrifuged for 5 minutes at 1000 rpm to obtain platelet-rich plasma. Part of the plasma is taken, and the remaining centrifuged at 3000 rpm for 20 minutes, receiving BeTP. BTP is standardized by the number of platelets by diluting the original BTP with an autologous BeTP sample (obtained before venous stasis - 1st test or after it - 2nd test) to 200 · 10 ⁹ / L. The concentration of blood platelets in the initial BTP is calculated in the Goryaev's chamber (in 50 large squares). The volumes of mixed BTP and BeTP are determined by the formula:

V _BeTP = V _BTP · [(N / 200000) -1],

where V _BeTP is the volume of platelet-poor plasma,

V _BTP - the volume of platelet-rich plasma,

N is the counted platelet concentration in the original BTP (cells / μl).

From a selected volume of standardized plasma, 0.02 ml of plasma is applied to a glass slide and 0.02 ml of a collagen inducer solution at a standard concentration with another pipette (1: 2 dilution of the main suspension). Plasma is mixed with inducers with a glass rod and the stopwatch is turned on. The mixture is stirred so that the liquid occupies a circle with a diameter of about 2 cm.

Shaking the glass in a circular motion in the transmitted light of the illuminator, on a black background they monitor the occurrence of aggregates through a magnifying glass. When distinct aggregates appear, the solution becomes clear and some of the aggregates adhere to the glass, the stopwatch is turned off, fixing the time of platelet aggregation.

The reaction is repeated 2-3 times in the 1st and 2nd samples, finding for each of them the arithmetic mean value.

The degree of inhibition of platelet aggregation after venous stasis is used to judge the anti-aggregation activity of blood plasma due to the entry of prostacyclin into the bloodstream from the vascular wall. The index of antiaggregatory activity of the vascular wall (IAASC) in the study of AT on glass was calculated by the formula:

The normal values for collagen can be considered IAASC 1.45-1.50.

The determination of the antithrombogenic properties of the vascular wall, depending on the production of antithrombin III activating substances, evaluating its basal level before and after temporary dosed ischemia of the vein wall, is given below (Barkagan Z.S., Momot A.P. Basics of the diagnosis of hemostasis disorders. M. , 1999 - 214 p.).

The principle of the method is based on the detection of a decrease in activity (in the absence of heparin) in antithrombin III (AT III) plasma, which is determined by the ability of the studied plasma to inactivate thrombin, for which it is pre-treated with a heparin sorbent, then subjected to thermal defibrination and mixed with a standard amount of thrombin. After incubation of the mixture, the residual coagulation activity of thrombin is determined in it. The level of decrease in thrombin activity as a percentage of the norm assesses the activity of AT III in the studied plasma.

The following reagents are used. 1. Tris-HCl buffer 0.05 M, pH 7.4. 2. Thrombin solution in buffer: 0.2 ml of the solution should coagulate with 0.3 ml of 0.4% fibrinogen solution for 15-16 seconds. The thrombin working solution is prepared in a plastic or silicone tube, stored at room temperature and used in the test on the day of the study. 3. Heparin sorbent Hepasorb-1 (company "Technology-Standard"). 4. Freshly prepared solution of fibrinogen at a concentration of 3.5-4.0 g / l, which is used to test the residual activity of thrombin. It is allowed to replace the fibrinogen solution with a normal platelet-poor citrate plasma diluted in buffer 2 times. 5. Fresh platelet-poor plasma obtained from 6-8 healthy piglets, or proprietary lyophilized, with known AT III activity, which is used to construct a calibration graph.

The material for the study is a citrate platelet-poor plasma.

To prepare sorbed and defibrinated plasma in a test tube, 10 mg of heparin sorbent is mixed with 1.0 ml of the studied plasma. Constantly shaking the tube, mix its contents at room temperature for 8 minutes, excluding the formation of foam and sediment at its bottom. After that, the mixture is centrifuged for 2 minutes at 1000-1500 rpm. The plasma in the supernatant is transferred to a clean tube and defibrinated in a water bath at + 56 ° C for 6 min, after which it is again centrifuged at 3000 rpm for 10 min, the supernatant is removed and examined for the first 2 hours.

The course of determination. 0.4 ml of a thrombin working solution is added to the test tube and heated in a water bath at + 37 ° C for 2 minutes. Then add 0.1 ml of the studied adsorbed and defibrified plasma, include a stopwatch. After 2 minutes (exactly!) 0.2 ml of the mixture was transferred to a test tube with 0.3 ml of fibrinogen solution (or diluted normal plasma), preheated to + 37 ° C. Immediately turn on the stopwatch and record the clotting time.

From the calibration curve, AT III activity is found in percent.

The construction of the calibration curve is as follows. The plasma of healthy people is defibrated as described above. Then, dilutions are prepared from it for constructing a calibration curve in accordance with Table 1.

Table 1 Scheme for preparing plasma dilutions for constructing a calibration curve in determining the activity of AT III Breeding number Plasma + buffer AT III activity,% one
2
3 Buffer free plasma
0.2 ml + 0.2 ml
0.1ml + 0.3ml one hundred
fifty
25

With each of the dilutions, the clotting time is determined by the above procedure. All measurements are carried out three times, the average result is calculated. When constructing a calibration curve along the logarithmic axis of ordinates, the clotting time in seconds is plotted, along the abscissa, the activity of AT III in% in accordance with the above dilutions. An approximate calibration curve is shown in the drawing.

A calibration curve for one type and one series of thrombin and buffer is built once. Only the activity of the thrombin working solution is periodically monitored.

Normally, without venous occlusion, AT III activity is 85-115%. When taking blood after temporary venous occlusion, as described in the method for assessing the antiplatelet activity of blood vessels and determining AT III in it, it is possible to indirectly assess the level of synthesis of tissue activators of AT III in the vessel wall. Against the background of temporary venous occlusion, the activity of AT III increases and is in the range of 120-143%. The vessel synthesis index of AT III activators (ISAAS) is calculated by dividing the value of AT III activity against venous occlusion by the value of AT III without it. Normally, it is 1.20-1.40.

The activity of the synthesis of tissue plasminogen activators by the time of lysis of the euglobulin clot before and after temporary venous occlusion was evaluated as follows.

The principle of the method is to precipitate the euglobulin fraction of plasma, while the main inhibitors of fibrinolysis, in particular α ₂ -antiplasmin, remain in the supernatant and are removed. Therefore, euglobulin lysis reflects the activity of fibrinolysis under conditions of exclusion of the inhibitory effect of antiplasmin. Its speed mainly reflects the amount of plasminogen in the plasma and the degree of its activation. The time of spontaneous lysis of a clot obtained from the euglobulin fraction of plasma is determined by adding a solution of calcium chloride to it.

The following reagents are used: 1% acetic acid solution. 2.2.277% solution of calcium chloride. 3. Michaelis buffer or Tris-HC buffer, 0.05 M, pH 7.3-7.4.

The material for the study is a citrate platelet-poor plasma before and after temporary venous occlusion. The course of determination. To obtain the euglobulin fraction, 0.15 ml of 1% acetic acid and 0.5 ml of the test plasma are added to 8.0 ml of distilled water. After 30 minutes of cooling the mixture in the refrigerator at + 2-8 ° C, the euglobulins are precipitated by centrifugation at 1500 rpm for 7 minutes. The supernatant is drained, the tube is dried by tipping over onto filter paper. The precipitate of euglobulins is diluted in 0.5 ml of buffer and placed in a water bath (+ 37 ° C). Then, without removing the tube from the bath, add 0.5 ml of calcium chloride solution, mix gently by shaking the tubes (without shaking!), Turn on the stopwatch and incubate in a water bath at + 37 ° C. Record the time from the moment of adding calcium chloride to the complete dissolution of the clot.

The result is expressed in minutes. Normally, the time of spontaneous lysis of the euglobulin clot is 180-240 minutes. After temporary venous occlusion, the lysis time decreases, being in the range of 126-170 minutes. When dividing the duration of spontaneous lysis of the euglobulin clot before occlusion by the value after, the index of fibrinolytic activity of the vascular wall (IFASS) was calculated, normally equal to 1.35-1.45.

For each laboratory, it is necessary to examine a group of healthy newborn piglets in order to find the average standard values with the calculation of S ² by all three methods for subsequent calculations.

For each method, for each newborn piglet with anemia, for greater diagnostic accuracy, a test is performed 3-4 times with the calculation of arithmetic mean values, which are used in subsequent calculations and the calculation of S ² .

To determine the weight of the individual studied parameters in vascular patients of piglets and to calculate the total antithrombotic potential of their vessels, a systematic multivariate analysis was used, which allows translating multidimensional quantitative characteristics of the studied process with incomparable absolute values into comparable relative values (Application of morphometry and statistical analysis methods in morphological studies. M.V. Uglova, B. A. Uglov, V. V. Arkhipov et al., Kuibyshev, 1982.- 47 p.). The calculation was made according to the formulas:

where X _j is the studied parameter, X ₀ is the normative parameter, X _j is the relative difference.

where Pi is the weight coefficient (coefficient of influence), a is a constant factor (in our studies a = 0.1), σ _j is the standard deviation of the value of X _j in relative units.

where S _i ² is the variance of the studied parameter X _i , n _i is the number of observations in determining X _i , S ₀ ² is the variance of the normative parameter X ₀ , n ₀ is the number of observations in determining X ₀ .

where X _{Bi OAPS} - a value that integrally characterizes the studied process at a given time (in relative units), is the total antithrombotic potential of blood vessels (OAPS).

The combined use of the described methods with the subsequent processing of the obtained IAASC, ISAAS, and IFASC values by systemic multivariate analysis with the determination of the total antithrombotic potential of blood vessels (X _{Bi OAPS} ) - the norm is 0.180 and higher, allows to identify the risk of weakening of antithrombotic activity of blood vessels in newborn piglets (X _{Bi OAPS} from 0.179 to 0.060), which requires normalization of feeding and keeping animals and the appointment of ferroglukin and monthly monitoring of the state of vascular functions. When the value of X _{Bi OAPS} from 0.059 and below - it is necessary to immediately treat newborn piglets according to the claimed method.

To correct the weakening of the functions of the vessel wall, newborn piglets with anemia are prescribed ferroglukin 150 mg intramuscularly, twice with an interval of 10 days, phosprenyl intramuscularly, three times in different syringes at different points, the first time 0.08 ml / kg simultaneously with the iron preparation , a second time at a dose of 0.07 ml / kg five days after the first injection of ferroglukin, a third time at a dose of 0.06 ml / kg simultaneously with a second injection of ferroglukin and gamavit 0.015 ml / kg intramuscularly once a day, eight days starting with the first injection of ferroglukin.

The introduction of this method of correcting the functions of the vessel wall in newborn piglets with anemia in the practice of pig farms will solve a number of pressing veterinary and economic problems of our time. Effective correction in newborn piglets with anemia will reduce the death rate in piglets, improve the herd, prevent the weakening of the animal organism, increase the weight gain and subsequently receive healthy offspring from these animals.

Example. In newborn piglet No. 26 with anemia, a violation of erythropoiesis and signs of a decrease in the level of iron in his body (serum iron 12.8 μmol / L, siderocytes 1.0%, with a hemoglobin quantity of 83.0 g / L, erythrocytes - 4.03 × 10 ¹² / l.

The pig was examined in a pigsty for an anemia clinic. Blood was taken from the piglet and examined according to the above scheme with the assessment of IAASC, ISAAS and IFAS and calculation of the total antithrombotic potential of blood vessels.

The animal showed pronounced signs of inhibition of the antithrombotic function of the vascular wall. IAASC was 1.07, ISAAS 1.10 and IFAS 1.14. The total antithrombotic potential of the vessels was X _{Bi OAPS} = 0.055.

The piglet was prescribed treatment: ferroglukin 150 mg (2 ml) intramuscularly, twice with an interval of 10 days, phosprenyl intramuscularly, three times in different syringes at different points, the first time 0.08 ml / kg simultaneously with the iron preparation, the second time at a dose of 0 , 07 ml / kg five days after the first injection of ferroglukin, the third time at a dose of 0.06 ml / kg simultaneously with the second injection of ferroglukin and gamavit 0.015 ml / kg intramuscularly once a day, eight days starting from the first injection of ferroglukin.

5 days after completion of treatment, the signs of anemia stopped - serum iron 19.9 μmol / L, siderocytes 7.9%, with a hemoglobin of 119.6 g / L, red blood cells - 4.97 × 10 ¹² / L), IAASC amounted to 1.50, ISAAS 1.37 and IFASS 1.42, normalization of the value of the total antithrombotic potential of blood vessels was noted (X _{Bi OAPS} = 0.182). Further observation of this pig did not reveal any deviations from the norm in the future, and the investigated parameters of the antithrombotic activity of the vessels remained within normal limits.

Using the proposed method for correcting the weakening of the vessel wall functions in newborn piglets with anemia in pig farming will help to avoid disturbances in their microcirculation and tissue trophism, slow growth and development of animals, vascular complications, improve the population, optimize weight gain and timely receive sufficient meat production.

Claims (1)

A method for correcting the weakening of the functions of the vessel wall in newborn piglets with anemia, characterized in that they administer 150 mg (2 ml) of ferroglukin intramuscularly, twice with an interval of 10 days, fosprenil intramuscularly, three times in different syringes at different points, the first time 0.08 ml / kg simultaneously with the introduction of ferroglukin, a second time at a dose of 0.07 ml / kg five days after the first injection of ferroglukin, a third time at a dose of 0.06 ml / kg simultaneously with a second injection of ferroglukin, and gamavit is additionally administered at a dose of 0.015 ml / kg intramuscularly once a day in for eight days, starting with the first injection of ferroglukin.

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