RU2308188C2 - Application of feedstuff treated with "hymizyme" polyenzymatic preparation in case of intoxication with heavy metals in animals and hens - Google Patents

Abstract

FIELD: veterinary science.

SUBSTANCE: it is necessary to apply the feedstuff treated with "Hymizyme" polyenzymatic preparation by high-temperature fermentation technique in case of intoxication with cadmium and lead in animals and hens. The innovation enables to decrease the content of heavy metals in meat and poultry eggs.

EFFECT: higher efficiency.

8 ex, 3 tbl

Description

The invention relates to the field of livestock with the possibility of its use in veterinary medicine, in particular to means of treatment and prophylaxis in case of excessive amounts in feed or poisoning of animals with heavy metals, namely cadmium and lead.

Of the large number of heavy metals of technogenic origin, the greatest danger is cadmium and lead, which enter the body of animals with air, water and feed.

One of the toxicity features of heavy metals is that they usually have a cumulative effect, i.e. able to accumulate in the body, and cadmium and lead are more endowed with these properties. The specificity of the toxic effect of heavy metals lies in the fact that, having a wide range of manifestations, these elements affect almost all organs and functional systems of the body, but the highest concentrations of cadmium and lead are observed in the liver and kidneys.

The excretion of heavy metals from the body occurs mainly with feces and, to a lesser extent, with urine, sweat, milk, saliva, bile and through the hairline, but the greatest danger is the amount of heavy metals that has not been eliminated from the body and passes from animal products to the body person.

Known means for treating animal poisoning with heavy metals are activated carbon, laxatives and enveloping agents (tannin, flaxseed decoction, pectin solution), unitiol (S. Dimitrov, A. Dzhurov, S. Antonov “Diagnosis of animal poisoning.” - M. : Agropromizdat, 1986; D.K. Chervyakov “Pharmacology with the formulation.” - M .: Agroproizdat, 1986), sodium sulfide (V.G.Sofronov, V.A. Novikov, V.A.Konyukhova, etc. - patent for the invention No. 2178701, M., 2002) and others. However, when ingested large doses of heavy metals, these drugs for the treatment of animals are not quite effective even with repeated use.

There is also known a tool for the treatment of cadmium and lead poisoning - sodium thiosulfate ("Veterinary Encyclopedic Dictionary". - M .: Soviet Encyclopedia, 1981; "Medicines" / Handbook. - M .: Russian book, 1994).

However, repeated intravenous administration of sodium thiosulfate in large doses during poisoning of animals with heavy metals significantly increases the complexity of veterinary specialists and limits its practical use in industrial animal husbandry, reduces the productivity of animals as a result of stress and side effects when fixing animals. In addition, sodium thiosulfate has a therapeutic effect in the first hours after poisoning, its use at a later date is ineffective.

The objective of the invention is the use of the multienzyme preparation "Gimizim", intended for the enzymatic treatment of concentrated feed, use for the prevention and treatment of poisoning of chickens with heavy metals. The prophylactic and therapeutic effects are manifested by feeding the fodder treated with the Gimizim multienzyme feed to poultry and animals.

The use of a multienzyme preparation for the prevention and treatment of heavy metal poisoning of chickens has great advantages over medicinal products in that it is used in animal feeding all the time, without negatively affecting the animal’s body and its products (meat and eggs), but rather contributing to only its increase, and in addition, additional funds are not spent on medications. In addition, the use of this drug will reduce the content of heavy metals in meat and eggs of birds grown in the technogenic regions of our country.

The mechanism of the protective action of fermented feed in case of heavy metal poisoning is obviously based on improving the metabolic processes of animals and birds, which in turn leads to an increase in immunity, which results in better survival in chronic poisoning of animals, which was especially evident in the first stages of the experiment, and subsequently, the correction of the adaptive reactions of the body begins to occur in response to the excess intake of cadmium and lead.

The Gimizim multi-enzyme preparation is produced by the Development of Economic Reforms Foundation (Kazan) and is designed for the enzymatic processing of concentrated feed at elevated temperatures into easily digestible animal feed. Gimizim contains a complex of hydrolytic enzymes providing, under recommended conditions, a quick and deep breakdown of food nutrients into low-molecular easily digestible forms, a complex of antioxidants, an adsorbent and a preservative. The drug is intended for high temperature fermentation of concentrated feed. The finished feed consists of 65-75% of water and 25-35% of grain fodder.

Feed preparation procedure:

1. Pour cold water into the feed mixer or other feed preparation tank.

2. Dissolve the drug "Gimizim" in 5-10 l of warm water (20-40 ° C) at the rate of 1 kg of the drug per 1000 kg of grain to be processed.

3. Add the dissolved preparation to the feed mixer with water and mix.

4. Pour coarse grinding fodder into the mixer.

5. Stir the moist mixer and heat to 70-100 ° С.

6. Leave to ripen for 1-2 hours.

The therapeutic and prophylactic efficacy of the Gimizima multienzyme preparation was tested on white rats and chickens during acute and chronic complex seeding of animals with cadmium and lead. Observation of animals was carried out throughout the experiment.

Example 1. The study of the preventive and therapeutic efficacy of the drug in experiments on rats. The experiments were carried out on white male rats with a live weight of 180-220 g, divided into two groups: the first served as a control, and the second as an experiment. Cadmium sulfate and lead acetic acid were orally administered to animals in an average lethal dose of LD ₅₀ - 350 mg / kg and 3000 mg / kg, respectively. Rats of the second group were fed grain fodder treated with the Gimizim polyenzyme preparation for a month before the experiment and throughout the experiment, and the first one that underwent all technological processing, but without enzymes.

In the study of animals, the general condition, attitude to feed and water, the condition of the mucous membranes and coat, body weight, the timing of death or recovery of animals were taken into account. The results of experiments to study the therapeutic and prophylactic effectiveness of the drug are shown in table 1.

Table 1
The therapeutic and prophylactic efficacy of the Gimizim multienzyme preparation for poisoning white rats with heavy metals at a dose of LD ₅₀ . Groups of animals Number of animals, goal Deadlines, days. In parentheses the number of dead animals Total survived fell 1. Seed with cadmium and lead thirty fifteen fifteen 1 (10), 2 (4), 3 (1) 2. Seed with cadmium and lead using fermented feed thirty 23 7 2 (2), 3 (4), 4 (1)

The table shows that in case of poisoning of white rats with cadmium sulfate and lead acetic acid at a dose of 350 mg / kg and 3000 mg / kg, respectively, the death of animals began on the 1st day after the administration of heavy metals, and continued for 3 days. A total of 15 white rats fell out of 30, the survival rate was 50%.

When laboratory animals were fed grain fodder treated with the Gimizim polyenzyme preparation, 23 out of 30 poisoned white rats survived, i.e. 76.7%. Moreover, in comparison with animals of the control group, clinical signs of poisoning developed later, namely on the 2nd day after the administration of heavy metals, and were less pronounced.

Symptoms of poisoning were characterized by excitement, refusal of food and water, ruffled coat, ataxia and weight loss. One rat (the first group) died 6 hours after drug administration. The condition of the surviving animals visually normalized during the first three to four days. In the next 14 days, no changes in the clinical condition of animals were noted.

Clinical examination of animals fed with grain fodder treated with Gimizim showed that weight loss and other signs of poisoning were less pronounced in rats of the control group. The surviving animals looked quite active, willingly took food and water.

Example 2. The study of the therapeutic and prophylactic effectiveness of the multienzyme preparation was carried out when feeding hens with grain fodder treated with Gimizim, when they were seeded with heavy metals. The birds were divided into two groups. Both groups of chickens were given cadmium sulfate and lead acetate at a dose of 52.5 mg / kg and 400 mg / kg live weight for 22 days in bread boluses, respectively, which is 1/10 LD _50, respectively. The first group served as control. Birds of the second group received grain fodder treated with the Gimizim polyenzyme preparation before and during the entire duration of the experiment, and the first one that underwent all technological processing, but without enzymes.

Determination of the clinical status of birds, as well as a blood test for the content of red blood cells, hemoglobin, white blood cells and white blood cell count, total protein and its fractions, cholesterol and sulfhydryl groups, as well as phagocytic activity, was carried out before the start of the seed (background) and on the 10th day control and experimental animals in comparative terms. All indicators were determined by conventional methods.

During the experiment, the content of heavy metals in the excreta of living, as well as the organs and tissues of dead and dead birds, which were determined on a Kvant-AFA atomic absorption spectrophotometer according to GOST 30178-96, using the Guidelines for the spectral determination of heavy metals in biological materials and environmental objects.

The results of experiments on the study of the therapeutic and prophylactic effectiveness of the drug for poisoning chickens with heavy metals at a dose of 1/10 LD ₅₀ are shown in table 2.

table 2
The therapeutic and prophylactic effectiveness of the Gimizim multienzyme preparation for daily seeding of chickens with heavy metals at a dose of 1/10 LD ₅₀ . Groups of animals Number of animals, goal Deadlines, days. In parentheses the number of dead animals Total fell counter. slaughter 1. Seed with cadmium and lead thirty 28 2 1 (8), 3 (9), 12 (10), 11 (11), 1 (12) 2. Seed with cadmium and lead using fermented feed thirty 28 2 2 (17), 3 (18), 5 (19), 8 (20), 9 (21), 1 (22)

The table shows that with daily seeding of chickens with cadmium sulfate and lead acetate at a dose of 52.5 mg / kg and 400.0 mg / kg, respectively, the death of the birds of the control group began on the 8th day after the introduction of heavy metals and continued for subsequent 5 days, and when feeding the birds fodder grain treated with the multi-enzyme preparation Gimizim, they began to die only on day 17. Moreover, compared with the birds of the control group, in which the clinical signs of poisoning in the form of general oppression, decreased appetite, ruffling began to develop on 3-4 days, in the experiment this happened at a later date, namely on the 5-6th day since the seed.

With the simultaneous introduction of lead and cadmium salts into the body at a dose of 1/10 LD ₅₀ on the 10th day of the study, the hemoglobin content in the control decreased to 73.5 g / l, which is 30% less than background data, red blood cells to 4.53 · 10 ¹² / l or 26%, and white blood cells up to 31.5 · 10 ⁹ / l or 10%, and in experimental chickens the content of red blood cells was 5.45 · 10 ¹² / l, white blood cells 33.2 · 109 / l and hemoglobin 90 , 3 g / l, which is lower than the background data, respectively, by 11%, 5.2% and 14%. In the structure of the leukoformula there is a statistically significant increase in basophils by 20% compared to the background, as well as a statistically unreliable increase in eosinophils. Throughout the experiment, a decrease in the total protein in blood serum is observed, for example, on the 10th day of the experiment in control chickens this indicator significantly differed from the background indicator and amounted to 43.1 g / l or decreased by 27%, while in experimental birds, these data were respectively 48.4 g / l and 18%. In the fractional composition of the protein in control birds, there was a significant decrease in the amount of albumin from 21.2 g / l at the beginning to 13.4 g / l on the 10th day of research, as well as a significant decrease in the level of globulin fractions. In experimental chickens, these indicators have changed slightly.

In control chickens poisoned with heavy metals at a dose of 1/10 LD ₅₀ , there was a slight decrease in phagocytic activity by 10% compared with background data on the 10th day from the start of the seed. In experienced chickens, this indicator has not changed much.

The number of sulfhydryl groups on the 10th day of the experiment decreased in hens of the control group from 2500 to 1825 mmol / 100 ml of blood or by 27%, and in the experimental group, respectively, to 2025 mmol / 100 ml of blood or by 19%. These changes were reliable.

Thus, studies have shown that with prolonged chronic poisoning with a mixture of cadmium and lead salts at doses equal to 1/10 LD ₅₀ , statistically significant changes occur in the leukoformula, the content of hemoglobin, total protein and protein fractions, phagocytic activity, as well as sulfhydryl blood groups , which were more pronounced in birds of the control group compared with experimental chickens.

In the study of heavy metals in the precipitates, it was found that the use of an enzyme preparation contributed to their better excretion from the body of birds, for example, the average daily content of cadmium and lead in the litter of birds in the experimental group was higher than in the control, respectively, 1.5 and 1.2 times. It should be noted that in the last 5–7 days of life, experimental heavy metal hens began to be excreted 2-3 times more with litter than in the first days of the experiment, for example, the average daily release of cadmium at the end of the experiment was 530–630 mg / kg of litter and at the beginning - 90-120 mg / kg, and lead, respectively, 930-1001 mg / kg and 100-130 mg / kg. A greater release of heavy metals with litter by the hens of the experimental group in comparison with the control indicates the positive effect of the feeds treated with the Gimizim polyenzyme preparation on the bird organism. The reason for the more efficient removal of heavy metals with litter in diets with fermented feed is probably in this case a deeper breakdown of feed nutrients into low molecular weight compounds, respectively, the number of functional groups able to bind heavy metals in comparison with non-hydrolyzed biopolymers is growing in the environment.

To study the content of heavy metals in organs and tissues on the 10th day of the experiment, a control slaughter of birds was performed, the results of which showed that the highest cadmium content was observed in the liver - 27.5 mg / kg in the experiment and 47.9 mg / kg in the control, stomach 6.97 mg / kg and 24.71 mg / kg in the kidneys, 40.2 mg / kg and 52.54 mg / kg, and lead in the stomach, 76.75 mg / kg and 145.96 mg / kg and liver - 32.1 mg / kg and 47.27 mg / kg, respectively. In the muscles, the cadmium content in the experimental chickens was 1.07 mg / kg and in the control 3.05 mg / kg, or 2.85 times less, and lead, respectively, 8.27 mg / kg and 12.0 mg / kg or 1 , 45 times less.

Heavy metals in organs and tissues were also studied in dead animals, as a result of which the highest cadmium content in the liver was also found - 56.9 mg / kg in the experiment and 62.6 mg / kg in the control, stomach - 46.5 mg / kg and 53.2 mg / kg in the kidneys - 43.56 mg / kg and 56.84 mg / kg, and lead in the stomach - 162 mg / kg and 186.5 mg / kg and liver - 114.6 mg / kg and 128.4 mg / kg, respectively. In the muscles, the cadmium content in the experimental hens was 1.57 mg / kg and in the control 3.1 mg / kg or 1.98 times less, and lead, respectively, 9.69 mg / kg and 12.78 mg / kg or 1 32 times less.

Thus, the results of the indication of salts of heavy metals in the organs and tissues of chickens during control slaughter and in the dead showed that the treatment of feed with a polyenzyme preparation contributed to a decrease in their deposition in the organs and tissues of experimental birds compared to the control.

Example 3. Given that, under natural conditions, it is possible that animals and birds are infected in smaller quantities, it was decided to reduce the dose of heavy metals introduced, therefore, keeping the principle of constructing an experiment similar to that described in Example 2, the hens of both groups (control and experiment) were asked for 120 days in bread boluses, cadmium sulfate and lead acetate, respectively, at a dose of 26.25 mg / kg and 200 mg / kg live weight, which is 1/20 LD ₅₀ .

Determination of the clinical status of birds, as well as a blood test for the content of red blood cells, hemoglobin, white blood cells and white blood cell count, total protein and its fractions, cholesterol and sulfhydryl groups, as well as phagocytic activity, was carried out before the start of the seed (background) and 10, 20, 30 , 60.90 and 120 days in control and experimental animals in comparative terms.

During the experiment, the content of heavy metals in the secretions, organs and tissues of dead and dead birds was taken into account.

The results of experiments on the study of the therapeutic and prophylactic effectiveness of the drug for poisoning chickens with heavy metals at a dose of 1/20 LD ₅₀ are shown in table 3.

Table 3
The therapeutic and prophylactic effectiveness of the Gimizim multienzyme preparation with daily seeding of chickens with heavy metals at a dose of 1/20 LD ₅₀ . Groups of animals Number of animals, goal Deadlines, days. In Brackets the number of dead animals Total counter. slaughter fell survived 1. Seed with cadmium and lead thirty four 26 - 1 (18), 2 (19), 4 (20), 10 (21), 6 (22), 1 (25), 1 (28), 1 (35) 2. Seed with cadmium and lead using fermented feed thirty 12 3 fifteen 1 (25), 1 (26), 1 (27)

From the table it follows that with daily seeding of chickens with cadmium sulfate and lead acetate at a dose of 26.25 mg / kg and 200.0 mg / kg, respectively (1/20 LD ₅₀ ), the death of control birds began on the 18th day after heavy metals and lasted for the next 18 days, and when feeding the birds fodder grain treated with the polyenzyme preparation Gimizim, they began to die only on day 25 and 3 heads died compared to the twenty-six in the control. Moreover, in comparison with the birds of the control group, in which the clinical signs of poisoning in the form of general inhibition, decreased appetite, ruffling began to develop on the 5-7th day, in the experiment this happened at a later date, namely on the 10-12th day since the introduction of heavy metals. In addition, it should be noted that in the hens of the experimental group, clinical signs of poisoning were observed within 2-3 weeks, during which 3 birds died, and in the surviving chickens the general condition and blood counts began to return to normal by 2-3 months from the beginning of the experiment , despite continued oral administration of heavy metals in a dose of 1/20 LD ₅₀ .

With the introduction of drugs in doses of 1/20 LD ₅₀ in control chickens, there was a slight decrease in hemoglobin in the blood. Moreover, on the 10th day this decrease was significant and amounted to 83 g / l, which is 21% less than the background value, the erythrocyte content also significantly decreased to 4.96 · 10 ¹² / l or 19%, and leukocytes, respectively, to 32.55 · 10 ⁹ / l or 7%. In experimental chickens, the content of erythrocytes was 5.75 · 10 ¹² , leukocytes 33.6 · 10 ⁹ and hemoglobin 99.7 g / l, which is 6%, 4% and 5.1% respectively lower than the background data. On the 10th day of the experiment, a statistically insignificant increase in basophils by 10% and eosinophils - 5% compared with background data is observed in the structure of the leukoformula of both groups.

Throughout the experiment, there has been a decrease in total protein in serum. On the 10th day of the experiment, this decrease in control chickens significantly differed from the baseline by 16% and amounted to 49.6 g / l, while in experimental hens this decrease was only 11% and was 52.5 g / l. In the fractional composition of the protein, a decrease in the amount of albumin is observed, which is reliable and differs from the background values in control birds by 24% and amounts to 16.1 g / l, and a significant decrease in the content of α-, β- and γ-globulins is also observed. The decrease in the level of protein fractions in experimental chickens was unreliable.

In control chickens poisoned with heavy metals at a dose of 1/20 LD ₅₀ , an unreliable decrease in phagocytic activity by 7% was observed compared with the background data on the 10th day from the start of the seed. In experienced chickens, this indicator remained at the same level.

The number of sulfhydryl groups on the 10th day of the experiment decreased in hens of the control group from 2500 to 2025 mmol / 100 ml of blood or 19%, and in the experimental group, respectively, to 2265 mmol / 100 ml of blood or 9.4%. These changes were reliable.

On the 20th day, all the studied parameters tended to decrease, both in the experiment and in the control, however, in the hens of the experimental group it was less pronounced compared to the control.

The general condition and the studied blood indices in the surviving hens of the experimental group began to return to normal by the 60-90th day, despite the continued oral administration of heavy metals in a dose of 1/20 LD ₅₀ .

Thus, studies have shown that with prolonged chronic seeding of cadmium and lead salts with hens at doses equal to 1/20 LD ₅₀ , statistically significant changes occurred in the content of hemoglobin, total protein and protein fractions, phagocytic activity, as well as sulfhydryl blood groups, which were more pronounced in birds of the control group compared with experimental.

When studying heavy metals in secretions, it was found that the use of an enzyme preparation contributed to their better excretion from the body of birds. So the average daily content of cadmium and lead in the litter of birds in the experimental group was lower than in the control, respectively, in 3.42 and 3.02 times. It should be noted that in the last 15 days of the experiment in experimental chickens, the average daily release of cadmium with litter was 23.81 mg / goal, and at the beginning of the experiment, 9.01 mg / goal or 2.6 times more than in the first days of the study and lead, respectively - 64.9 mg / goal and 20.85 mg / goal, or 3.11 times. A greater release of heavy metals with the litter of hens from the experimental group, compared with the control, and especially in the last days of the experiment, indicates a positive therapeutic and prophylactic effect of feeds treated with the Gimizim polyenzyme preparation on the bird organism.

To study the content of heavy metals in organs and tissues, a control slaughter of birds was performed on the 10th day of the experiment, the results of which showed that treating feed with a polyenzyme preparation reduced the deposition of heavy metals in the organs and tissues of experimental animals. In particular, the highest cadmium content is observed in the liver - 19.27 mg / kg in the experiment and 31.98 mg / kg in the control, kidneys - 33.64 mg / kg and 34.52 mg / kg and the stomach - 3.3 mg / kg and 7.45 mg / kg, and lead in the stomach - 41.14 mg / kg and 93.14 mg / kg, kidneys - 33.64 mg / kg and 42.66 mg / kg and liver - 27.61 mg / kg and 39.84 mg / kg, respectively. In the muscles, the cadmium content in experimental chickens was 0.415 mg / kg and in the control 1.25 mg / kg or 3 times less, and lead - 5.14 and 8.87 mg / kg or 1.72 times less. On the 20th day of the experiment, the control slaughter was again performed, which showed that the content of heavy metals in the organs and tissues increased both in the experiment and in the control, however, the hens of the experimental group had cadmium and lead in the organs and tissues less than in the control: in the liver, cadmium was 1.5 times and lead 1.4 times, in the kidneys 5% and 25%, in the stomach 1.9 and 2.1 times, respectively.

The content of heavy metals in organs and tissues was also studied in dead animals, as a result of which the highest amount of cadmium was found in the liver - 57.1 mg / kg in the experiment and 63.46 mg / kg in the control, in the muscle tissue of the stomach - 43, 5 mg / kg and 55.18 mg / kg in the kidneys - 39.45 mg / kg and 60.18 mg / kg, and lead in the stomach - 159.21 mg / kg and 192.16 mg / kg and liver - 105 35 mg / kg and 119.21 mg / kg, respectively. In muscle tissue, the average cadmium content in experimental chickens was 1.51 mg / kg and control 2.95 mg / kg or 1.95 times less, and lead, respectively, 9.91 mg / kg and 14.62 mg / kg or in the experiment, 1.47 times less than in the control.

Thus, we can conclude that the treatment of feed with the polyenzyme preparation Gimizim contributed to a decrease in the concentration of heavy metals in the organs and tissues of experimental birds compared to the control.

When studying the content of heavy metals in eggs, the following conclusion was made: in control chickens, the average cadmium content was 0.462 mg / kg, and in experimental chickens 0.260 mg / kg, and lead, respectively, 6.66 mg / kg and 3.967 mg / kg, therefore, the use of an enzyme preparation in chickens of the experimental group made it possible to reduce the cadmium content in eggs by 1.78 times and lead by 1.68 times in comparison with the control.

The mechanism of the protective action of fermented feed in case of heavy metal poisoning is obviously based on the following factors:

firstly, as noted above, the content of the intestine of the bird has higher complexing (binding) properties with respect to heavy metal ions due to an increase in the number of low molecular weight compounds with functional groups compared to only partially hydrolyzed biopolymer;

secondly, an increase in feed biogenicity after fermentation leads to an increase in the body’s immunity and protective properties, thanks to this, animals and poultry more easily tolerate the initial period of acute poisoning, after which the body manages to start developing additional protection factors against toxicants (an increase in the content of sulfhydryl groups in the experiment compared with control);

thirdly, the detoxification of feed during high-temperature fermentation (decomposition of anti-nutritional substances, mycotoxins, etc.) leads to the focusing of the body's protective properties on the fight against only one toxicant - heavy metals, and accordingly a more effective body defense is obtained;

fourthly, in the birds of the experimental group, under the influence of feeds treated with the Gimizim polyenzyme preparation, the correction of the organism’s adaptive reactions probably begins to occur in response to the excess intake of cadmium and lead.

Thus, the studies showed that the use of feeds treated with the Gimizim polyenzyme preparation, unlike existing drugs, will save on drug costs, facilitate the work of veterinarians, will not create unnecessary stressful situations and, at the same time, will not occur the need to diagnose poisoning of animals and birds that time is wasted by laboratory staff and reagents, in addition, the use of this drug will reduce the content yellow metals in meat and eggs of birds grown in industrial regions of the country with their high concentration.

Claims (1)

The use of feed treated with the Gimizim polyenzyme preparation using high-temperature fermentation technology as a therapeutic and prophylactic agent in case of excessive content in feeds or poisoning of animals and chickens with heavy metals - cadmium and lead.