INTRODUCTION:

Relevance. The measures for fighting bacterial infections include a set of veterinary and sanitary measures, where an important role is played by the use of antibacterial drugs, including antibiotics^1-3. In industrial poultry breeding, antibiotics have been widely used for the treatment and prevention of infectious diseases for decades. This naturally resulted in the transfer of antibiotic resistance from the strains of animal microorganisms to the microorganisms of the human population, resulting in a reduction of the range for choosing antibiotics for humans. The cumulative properties of these drugs, contributing to their accumulation in the organism, and, therefore, in poultry meat, cause allergies in humans, and primarily in children^{4 - 5}.

Recent years have seen the trend to the development and use of exclusively veterinary antibiotics not used in medicine. This greatly reduces the risk to human health by reducing the opportunities for the development of cross-resistance to microorganisms that are potentially hazardous both for animals and for humans. However, it does not eliminate the problem of removing the accumulated antibiotic from the organism. The time of antibiotics removal from the animal organism is an important indicator of efficient production. An important task of pharmacology that requires using all methods available is speeding up the removal of drugs from the organism without reducing their antimicrobial efficacy.

One of these methods is the use of special absorbing and antioxidant feed additives that accelerate the removal of the drug many times after its administration is stopped^{6 - 10}.

The Reasil® Humic Health feed additive is exactly the product of this kind. Due to its active substance that consists of humic and fulvic acids, it helps remove antibiotics from the organism, resulting in increased product safety and the possibility to use it faster due to the reduced period of the antibiotic decomposition in the organism. All this happens because Reasil® Humic Health saves the resources of the organism using the chemical composition of humic acids, rather than the specific enzymes in the organism.

METHODS:

During the study, two experiments were performed based on the Slavinskaya poultry factory in Slavyansk-on-Kuban, Krasnodar Krai, and at the in-patient facility of the Saratov State Vavilov Agrarian University, Saratov.

For the first experiment at the Slavinskaya poultry factory in February 2019, four buildings with broiler chickens were chosen. Building No. 22 with a population of 38.788 thousand broiler chickens and building No. 16 with a population of 40.04 thousand broiler chickens were the reference, and building No. 23 with a population of 38.8 thousand broiler chickens and building No. 20 with a population of 45.31 thousand broiler chickens were experimental. The broiler chickens in the reference groups received the PK-6-1-1225 premix in the combined feed; and those of the experimental groups-the PK-6-1-652 premix with the Reasil® Humic Health feed additive in the dosage of 0.5 kg per one ton of combined feed. The composition of the combined feed is shown in Table 1.

Table 1. Combined feed composition, %

Name	Group
Name	Reference	Experimental
Wheat	18.00	18.00
Maize	42.22	42.27
Soybean cake	10.20	10.20
Soybean meal (CP 46 %)	11.50	11.50
The sunflower meal (CP 36 %, CF 21 %)	3.60	3.60
Sunflower meal (CP 36 %, CF 19 %)	5.30	5.30
Maize gluten	2.00	2.00
Sunflower oil	3.15	3.15
Lysine sulfate (70 %)	0.66	0.66
Dl-methionine	0.29	0.29
L-threonine 98 %	0.17	0.17
Table salt	0.28	0.28
Monocalcium phosphate	0.79	0.79
Limestone	1.13	1.13
Reasil® Humic Health	-	0.05
Natural sodium sulphate	0.10	0.10
Potassium carbonate	0.20	0.20
Vitamin B₄ (60 %)	0.06	0.06
Premix	0.20	0.20
Ovocrack	0.05	0.05
Poulcox (40 %)	0.05	0.05

The schemes of preventive treatment for the broiler chickens in the reference and experimental groups were identical, as shown in Table 2.

In the second experiment on the basis of the in-patient facility of the Saratov State Vavilov Agrarian University, the authors studied the effect of the dry feed additive based on unmodified microporous humic acids extracted from leonardite with the content of humic acids exceeding 80.0 % of the dry substance on the rate of the Florfenicol antibacterial drug removal from the organisms of broiler chickens.

Twenty-one-days-old broiler chickens of cross COBB-500 were used for the experiment. All broiler chickens were kept in the same room, under the same conditions optimal for this species. The broiler chickens were kept in cages 15 broiler chickens in each, and were identified with tags. Before the experiment, all cages were labeled. The broiler chickens in the experimental groups were fed with the Reasil® Humic Health feed additive based on humic acids. The scheme of the experiment is shown in Table 3.

Table 3. The scheme of the second experiment

Group	Experiment variant	Dosage of Reasil® Humic Health per 1 kg of feed	Method of introducing Florfenicol
Reference 1	Main diet (MD) + Florfenicol (FF)	-	Orally for seven days
Experimental 2	MD + FF + Reasil® Humic Health (RHH)	1.0 g	Orally for seven days
Experimental 3	MD + FF + RHH	1.5 g	Orally for seven days
Experimental 4	MD + FF + RHH	2.0 g	Orally for seven days

The broiler chickens were chosen for the study if the following conditions were met:–the broiler chickens felt good and had no pathological deviations;–no drugs were used for feeding them within at least 21 days before the start of the experiment; and–the broiler chickens had a balanced temperament and were not dangerous to the personnel.

For taking biological material, the broiler chickens were euthanized. After that, 1x1 cm pieces of the thigh and pectoral muscles in equal amounts were taken. The muscle tissue was packed in containers and stored at -20 ⁰C until laboratory tests.

The concentration of Florfenicol was determined by the ELISA method using the MaxSignal® reagent kit from BIOO Scientific Corporation (BIOO). The studies were performed on a Multiskan Go immunoassay analyzer for spectroscopy in the visible region for microtitration plates.

The biological material was taken 24 hours after the antibiotic had been given with drink, and 24 hours, 96 hours, seven days, and 10 days after the end of giving the antibiotic with water.

The method of administering the Florfenicol drug and the dosage were chosen following the instructions for use.

The dosage of Florfenicol for the broiler chickens was 100 ml/100 l of water administered to a group up to four weeks of age, and 200 ml/100 l of water in other age categories (20 mg/kg of the chicken weight). The drug was administered orally with water once a day for seven days.

Humic acids were used together with the antimicrobial drug orally with the feed throughout the entire experiment.

The criteria for assessing the efficiency of the Reasil® Humic Health feed additives based on humic acids were the speed of removing the Florfenicol antibiotic after stopping its administration to the broiler chickens, and preservation of the therapeutic concentrations of the antimicrobial drug, when it was used together with the Reasil® Humic Health feed additive.

The safety of using the drug in this kind of study was determined by the presence or absence of side effects, complications, and adverse (allergic) reactions in the broiler chickens after administering the drugs.

The obtained results were statistically processed by the dynamics of the body weight gain, the changes of blood serum hematological and biochemical parameters according to the standard procedures in Microsoft Excel 2016 (Microsoft Corp. USA) and the StatPlus 2009 Professional 5.8.4 statistical data analysis package for Windows (StatSoft Inc., USA) using Student's t-test for assessing the significance of differences between the samples for the experimental and reference groups. By the results of calculating the arithmetic mean and the standard deviation for this sample, the standard error of the arithmetic mean and the boundaries of its confidence interval were determined with regard to Student's coefficient t (n, p) with the significance level of 95 % (p = 0.05) and the number of measurements n = 3.

The reliability of the differences between the average values in the experimental and reference experiments was assessed by the magnitude of the p-value in the two-sample unpaired t-test with unequal dispersions.

The differences of the parameters were considered veracious with the inequality p ≥ 0.05. Besides, compliance with inequality t, t (n, p) was also monitored in these cases with n = (df + 1) (where df was the number of degrees of freedom), p = 0.05, where t = |x1–x2| / (s12 + s22)1/2, x1 and x2 were the arithmetic means, and s1 and s2 were their standard errors for the two samples of the experimental data. The differences were determined at the level of significance P ≤ 0.05.

The digital material was presented in SI units as recommended by the World Health Organization and the CMEA 1062-78 standard.

RESULTS:

In the first experiment, the broiler chickens were put in building No. 16 (reference) and building No. 20 (experimental) on 04.02.2019 and grown until 14.03.2019. The broiler chickens were placed in building No. 22 (reference) and building No. 23 (experimental) on 05.02.2019 and grown until 15.03.2019. The results are shown in Table 4.

At the age of 18 days, 10 broiler chickens from each group were check-slaughtered, and their muscle tissues were studied for the presence of the residues of antibiotics and anticoccidial drugs. It is known that the period of removing trimethoprim, sulfadiazine, and doxycycline from the organism is seven days, oxytetracycline — 14 days, and gentamycin — 21 days. Based on the scheme of therapeutic prevention in the broiler chickens in the experimental group, it may be assumed that oxytetracycline and gentamycin should be present in the muscle tissue of the broiler chickens at this age.

Table 4. Production performance of growing the broiler chickens

Group	Building No. 16	Building No. 23	Building No. 22	Building No. 20
The average initial weight of broiler chickens, g	40.00	33.00	33.00	38.00
Preservation rate, %	94.30	95.10	95.40	97.50
Sent to be used as Cornish chickens, specimens	0	1,000	0	1,000
Keeping period, days	36.60	36.10	35.90	35.60
The average weight of a single broiler chicken, kg	2.287	2.155	2.136	2.245
Net gross weight gain, kg	84,719	77,288	77,741	96,485
Average daily weight gain, g	61.10	58.70	58.60	62.50
Feed conversion	1.62	1.62	1.62	1.62

The result of the studies for detecting the presence of the residues of antibiotics and anticoccidial drugs in the meat of the broiler chickens in the reference group with the "Premi test" system showed the positive result (Fig.1).

Figure 1. The result of the analysis for the presence of residual quantities of antibiotics and anticoccidial drug in the meat of the broiler chickens in the reference group with the "Premi test" system – positive

The result of the studies for detecting the presence of the residues of antibiotics and anticoccidial drugs in the meat of the broiler chickens in the experimental group with the "Premi test" system showed the negative result (Fig. 2). This is evidence of the fact that the Reasil® Humic Health feed additive speeds up the process of removing antibiotics and anticoccidial drugs from the muscle tissue of the broiler chickens.

Figure 2. The result of the analysis for the presence of residual quantities of antibiotics and anticoccidial drug in the meat of chickens in the experimental group with the "Premi test" system – negative

The repeated tests for the presence of residues of antibiotics and anticoccidial drugs in the meat of chickens at the age of 21 days showed the same results. In this regard, the chickens from the reference buildings were not delivered as Cornish chickens. The absence of antibiotics and the anticoccidial drug in the muscle tissues of the broiler chickens in the experimental groups allowed slaughtering 1.0 thousand chickens to be used as Cornish from each of the buildings No. 20 and No. 23.

Slaughtering and anatomical dissection of 10 physiologically healthy chickens from each reference building at the age of 21 days showed the presence of fatty change of liver in 15 % of the studied chickens. At the same time, studying the liver of 1,000 chickens from each experimental building slaughtered to be used as Cornish showed that 100 % of the studied liver was normal.

Throughout the experiment, no clinical changes in the overall state and behavior were observed during daily inspections of all groups of chickens, nor were there any violations of motor activity, appetite, and water consumption.

The results of the studies showed that during the experiment, no external symptoms of intoxication had been observed in chickens. All chickens in both the experimental and the reference groups were active. The reaction to external stimuli was preserved. The broiler chickens' body temperature throughout the experiment remained within the physiological norms in all groups of chickens.

The effect of joint oral administration of the Florfenicol antibacterial drug and humic acid on the apparent concentration of the antibiotic in the organism during its use and the rate of removal after its termination was assessed by Florfenicol concentration in the muscle tissues. The results are shown in Table 5.

The studies showed that on the first day of the experiment, directly after the administration of the antibiotic without using the Reasil® Humic Health feed additive, its concentration in the muscle tissues amounted to 1,889.96 ± 368.78 ng/g of the biological material. This concentration was sufficient for the manifestation of the therapeutic effect in the chicken organism.

Seven days after the joint administration of the antibacterial drug and the Reasil® Humic Health feed additive and 24 hours after the cessation of the antibiotic in all groups of chickens, no changes were noted in the concentration of the antibacterial drug in the muscle tissues. Thus, it may be concluded that the joint use of the Florfenicol antibiotic and the Reasil® Humic Health feed additive does not have a negative effect on the apparent therapeutic concentration of the antibiotic in the chicken organism. No veracious differences were noted between the groups of chickens.

72 hours after the cessation of the antibacterial drug, a statistically veracious reduction of Florfenicol in the muscle tissues of the chicken, both in the experimental and the reference groups, to 96.64 ± 23.26; 46.26 ± 12.6; 23.72 ± 5.7, and 27.43 ± 6.6 ng/g of the muscle tissues, respectively, was noted. However, it should also be noted that more intensive removal of the antibiotic from the organism was observed in the experimental groups of chickens. For instance, in experimental group 1, its concentration was two times lower than in the reference, and in groups 3 and 4-four times lower. The content of the antibacterial drug in the muscle tissues in groups 3 and 4 showed no veracious difference.

Along with that, given the fact that in the Russian Federation and the EEU countries, the maximum allowed levels of Florfenicol in food products are regulated by the "Uniform Sanitary and Epidemiological and Hygienic Requirements for Goods Subject to Sanitary and Epidemiological Supervision (Control)", and in the meat of cattle and small cattle it should not exceed 0.2 mg/kg, in the meat of pigs-0.3 mg/kg, and in the poultry meat-0.1 mg/kg or 100 ng/g, it may be concluded that in the reference group of chickens that did not receive the Reasil® Humic Health feed additive, the average concentration of the antibiotic was at the maximum allowed level. With that, Florfenicol concentration in the muscle tissues of about one-third of the broiler chickens was above the norm. At the same time, in the experimental groups, its content was two and four times lower, and veraciously lower than the maximum allowed concentrations.

Seven and 10 days after the cessation of the antibacterial drug, trace concentrations of the antibacterial drug in the muscle tissues of chickens were observed in both the experimental and the reference groups. No veracious differences were noted between the groups.

During the administration of the antibiotic, the Reasil® Humic Health feed additive, remaining in the organism, did not remove the antibiotic, and, therefore, did not interfere with its effect. Removal of the antibiotic started in two days after the cessation of administration with water. The Reasil® Humic Health feed additive accelerates this process two times with the administration in the dosage of 1.0 g/kg of the feed. Further increasing the dosage of the Reasil® Humic Health feed additive to 1.5 and 2.0 g/kg of the feed accelerates the removal of the antibiotic from the muscular tissues two or four times, compared to the reference.

The obtained results may be explained by the fact that the mechanism of humic acids' action on a living organism tends to their antioxidant action. The high catalytic activity of humic acids was noted in the disproportionation of superoxide. This mechanism is most likely associated with high content of aromatic fragments and free radicals, which are suppliers of hydrogen ions for the enzymatic reactions caused by the presence of the fragments of semiquinone. These are capable of interacting with peroxide compounds, the excessive formation of which, in various diseases, accompanied by inflammatory and destructive processes, contributes to the destruction of cell membranes, resulting in cell death.

Because the metabolism of Florfenicol proceeds with the participation of the system of the microsomal fraction of hepatocytes, monooxygenases, cytochromes of the C reductase, and the P-450 cytochrome, the universal cofactor in these systems is restored NADPH. Florfenicol biotransformation is the oxidation of glutathione with subsequent conjugation of metabolites with endogenous molecules that actively transport it and excrete the biotransformed products in bile and urine.

Florfenicol biotransformation leads to the natural depletion of glutathione. At the same time, in the groups of chickens where the preparation of humic acids was used that had antioxidant properties and with high probability could bind the metabolites of Florfenicol, the depletion of glutathione in hepatocytes in response to exposure to the xenobiotic was avoided. All this promotes the acceleration of biotransformation and, consequently, more active removal of the antibiotic from the organism of chickens.

DISCUSSION:

The antibacterial activity of various antibiotic drugs, including Florfenicol, has been studied and proven in several experiments with farm animals and poultry^{2
- 3}. As pure Florfenicol is virtually insoluble in water, various adjuvants are used, which ensure good solubility of the drug, and maintain the stability of its aqueous solutions^{3, 10}. One of the main criteria for assessing the efficiency of both the antibacterial drug itself and the adjuvants used for improving its effect on a living organism is the rate of maintaining the therapeutic concentration of the drug and the rate of removing the antibiotic after the cessation of its administration with water.

Exactly these results were obtained in the studies of a sufficiently large population of poultry in the conditions of the Slavinskaya poultry farm in Krasnodar Krai and the veterinary in-patient facility of the Saratov State Vavilov Agrarian University in Saratov. It has been found experimentally that the Reasil® Humic Health feed additive in the dosage of 0.5 g/kg removes antibiotics and anticoccidial drugs from the muscle tissues of the broiler chickens by the age of 18 days, thereby allowing selling them as Cornish chickens. The results of studying the liver of 1,000 chickens slaughtered for Cornish chickens from every experimental building of the poultry farm show the absence of a fatty change of liver.

The joint use of the Florfenicol antibiotic and the Reasil® Humic Health feed additive does not prevent the accumulation of antibacterial drugs in the muscle tissues in therapeutic concentrations, and only 72 hours after the administration with water is stopped, the antibiotic is actively removed from the organism.

Veterinary supervision of residual quantities of drugs in the products of animal breeding is of great importance for obtaining environmentally pure products free from antibiotics. These issues are subject to study by both veterinary services and veterinary and sanitary experts^{1, 11, 12}.

The obtained results about the rate of removing the antibacterial drug confirm the fact that the preparation of humic acids can bind metabolites of Florfenicol and two to four times accelerate the removal of the antibacterial drug from the organism of chickens, depending on the dosage of the Reasil® Humic Health feed additive.

CONCLUSION:

As a result of the research, it has been found that the combined use of the Florfenicol antibiotic and the Reasil® Humic Health feed additive based on humic acids does not prevent the accumulation of the antibacterial drug in the muscle tissues in therapeutic concentrations. The joint use of the Florfenicol antibiotic and the Reasil® Humic Health feed additive based on humic acids in the dosage of 1.0 g/kg of the feed accelerates two times, and in the dosage of 1.5 and 2.0 g/kg of the feed, accelerates four times the removal of the antibacterial drug from the chicken organism after the cessation of the antimicrobial therapy.

REFERENCES:

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2. Plomodyalov D. Effektivnost florfenikola v lechenii tsyplyat [Efficiency of Florfenicol in the treatment of chickens]. Animal Breeding in Russia. 2019; 10: 26–27.

3. Khmyrov AV and Gorshkov GI. Perenosimost, farmakokineticheskie pokazateli i terapevticheskaya effektivnost Florama v opytakh na tsyplyatakh [Toleration, pharmacokinetic parameters and therapeutic efficacy of Floram in the experiments with the chickens]. Bulletin of the Kursk State Agricultural Academy. 2014; 9: 69–71.

4. Borisenkova AN, Novikova OB and Okonevsky P. Florfenikol v ptitsevodstve [Florfenicol in poultry breeding]. Poultry Breeding. 2012; 3: 43–45.

5. Tsyganova SV. Problema salmonelleza ptits-prepyatstvie dlya polucheniya biobezopasnykh produktov [The problem of avian salmonellosis is an obstacle for obtaining biologically safe products]. Poultry breeding. 2014; 4: 43–47.

6. Shermer CL, Maciorowski KG, Bailey CA and Byers FM. Caecal metabolites and microbial populations in chickens consuming diets containing f mined humate compound. Journal of the Science of Food and Agriculture. 1998; 77: 479–86.

7. Dolgopolov VN. Opyt primeneniya Gumivala dlya uluchsheniya produktivnosti krupnogo rogatogo skota, svinei i ptitsy [Experience in using Humival for improving the productivity of cattle, pigs, and poultry]. In Results and perspectives of using humic substances in productive farming and horse and poultry breeding: a collection of reports for conference, Edited by Berkovich AM. RANS, FGOU VPO "Moscow State Academy of Veterinary Medicine and Biotechnology named after K.I. Scriabin", Ministry of Agriculture and food of the Moscow Region, Pharmaceutical enterprise "Ligafarm", Moscow. 2006; pp. 40–43.

8. Vasiliev AA. Znachenie, teoriya i praktika ispolzovaniya guminovykh kislot v zhivotnovodstve [Importance, theory, and practice of using humic acids in animal breeding]. Agricultural Scientific Journal. 2018; 1: 3–6.

9. Vasiliev AA, Korsakov KV, Moskalenko SP, Kuznetsov MY, Sivokhina LA, Kitaev IA and Manieson VE. Kormovye dobavki na osnove guminovykh kislot iz Leonardita protiv mikotoksinov [Feed additives based on humic acids extracted from lignite for the treatment of mycotoxins]. Forage production. 2018; 5: 33–37.

10. Voronina IA, Garanicheva AV, Oborin AE, Gavrikov AV, Liepa VL and Khairetdinova MN. Vliyanie vspomogatelnykh veshchestv i rastvoritelei na toksichnost preparatov na osnove florfenikola [The effect of adjuvants and solvents on the toxicity of drugs based on Florfenicol]. Veterinary Medicine. 2012; 1: 52–54.

11. Shabunin SV, Vostroilova GA and Bliznetsova GN. Veterinarnyi kontrol za ostatochnymi kolichestvami lekarstvennykh sredstv v produktsii zhivotnovodstva na primere preparatov na osnove tsiminalya [Veterinary monitoring of residual quantities of drugs in animal breeding products on the example of drugs based on Ciminalum]. Achievements of science and technology in agriculture. 2012; 1: 21–22.

12. Korsakov KV, Vasiliev AA, Moskalenko SP, Sivokhina LA and Kuznetsov MY. Primenenie kormovykh dobavok s guminovymi kislotami v ptitsevodstve [The use of feed additives with humic acids in poultry breeding]. Zootechny. 2018; 4: 11–13.

Received on 23.01.2020 Modified on 13.03.2020

Research J. Pharm. and Tech. 2020; 13(12):6113-6119.

DOI: 10.5958/0974-360X.2020.01066.5

Day of growing	Vitamin and mineral additives	Antibiotics		Vaccinations
Day of growing	Vitamin and mineral additives	method of use	active substance	Vaccinations
day 0	Ascorbic acid	Aerosol	Gentamycin
day 1 – 3		Aerosol	Doxycycline
day 4				IB 4-91
day 6 – 8			Oxytetracycline
			Trimethoprim
			Sulfadiazine
day 9	Mineral complex			Infectious bursal disease (IBD)
day 10	Mineral complex
day 10 – 11	Calcium program
day 13	Vitamin complex			Newcastle disease (ND)
day 14	Ascorbic acid
day 15	Ascorbic acid			IB
day 16	Ascorbic acid
day 17	Citric acid
day 18	Ascorbic acid

Group	The period of the experiment
Group	During antibiotic administration with drinking	24 hours after the end of the antibiotic administration with drinking	72 hours after the end of the antibiotic administration with drinking	7 days after the end of the antibiotic administration with drinking	10 days after the end of the antibiotic administration with drinking
Reference	1,889.96 ± 368.78	1,583 ± 381.04	96.64 ± 23.26	4.84 ± 0.16	2.97 ± 0.19
Experiment 1	1,889.96 ± 368.79	1,765 ± 352.8	46.26 ± 12.6*	4.57 ± 1.39	2.92 ± 0.17
Experiment 2	1,889.96 ± 368.80	1,587 ± 382.17	23.72 ± 5.7**	4.32 ± 0.52	2.34 ± 0.55
Experiment 3	1,889.96 ± 368.81	1,780 ± 428.64	27.43 ± 6.6**	4.88 ± 0.58	2.33 ± 0.27