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Сельскохозяйственная биология, 2010, № 2, с. 71-76.

УДК [636.92+636.598]:636.08.003:636.087.8

NONSPECIFIC RESISTANCE AND PRODUCTIVITY IN RABBITS AND GOOSES UNDER THE INFLUENCE OF LIVE AND DEAD CULTURE OF Lactobacillus amylovorus

B.V. Tarakanov¹, T.A. Nikolicheva¹, L.L. Polyakova¹, V.V. Gerasimenko², V.N. Nikulin²

The authors estimated the effect of feeding of live and dead culture of Lactobacillus amylovorus on nonspecific resistance and productivity of growing rabbits and gooses. It was revealed, that both inactivated preparation of lactobacilli and live preparation stimulate humoral immunity and promote to the raise of productivity.

Key words: rabbits, gooses, feeding, Lactobacillus amylovorus, inactivation, unspecified resistance, growth rate.

 

The development and functions of the immune system are greatly dependant on the intestinal microflora. The data obtained from the model of gnotobiotic animals with underdeveloped lymphoid organs, low phagocytic activity of cells of the reticuloendothelial system, reduced concentration in serum of immunoglobulins of all classes and with deficit in the intestine of immunoglobulin-synthesizing cells, suggest that these anomalies disappear after the colonization in gastrointestinal tract of normal microflora (1). However, the immunostimulatory influence of its various representatives is a poorly studied matter. The immunomodulatory effect was established for live and dead  cultures of Bifidobacterium, Lactobacillus and Enterococcus. Their intragastral introduction to mice stimulated the increase in number of immunoglobulin-synthesizing cells in lamina propria of the small intestine and improved resistance to experimental Salmonella infection. (2).
The purpose of this work was studying the influence of live and dead cultures of Lactobacillus amylovorus the strain BT-24/88 (B-6253) on the immune system and productivity of rabbits and gooses.
Methods. The experiment was carried out in vivarium of the Institute on rabbits of the California breed. Five groups of 5-month-old rabbits (5 in each group) were formed by the method of pairs-analogues. The average live weight of one rabbit - 2200 - 2300 g; the animals were kept separately in mesh cages. The basic diet (BD) consisted of 200 g cereal hay and 200 g mixed fodder. 100 g mixed fodder contained 0,115 food units, 1.2 MJ metabolizable energy, 16.3 g crude protein, 4.9 g crude fiber, 0.7 g calcium and 0.5 g phosphorus.
Every day for 2 months, the control group I was fed with BD only. The other rabbits were given the diets containing BD and additives: control group II - 5 ml of culture medium for cultivation of L. amylovorus the strain BT-24/88; experimental group III - 1 ml of live culture (9,5 x 109 CFU / ml) and 4 ml of culture medium; experimental group IV - 1 ml of dead (1 atm., 60 min) culture and 4 ml of culture medium ; experimental group V - 5 ml of dead culture.
The animals were weighed at the start of the experiment, the feed consumption was estimated at the end of the 1 st and 2 nd months. At the end of the experiment, 3 rabbits from each group were slaughtered by decapitation; blood (by dissection of neck vessels) and urine samples (directly from the bladder after opening the abdominal cavity) were collected. The authors examined anatomopathological condition of organs and tissues, and weight of the liver, kidneys, heart, lungs, spleen, thymus and adrenal glands.
The blood was tested on the number of red blood cells, white blood cells formula (WBC), erythrocyte sedimentation rate (ESR) - by micromethod of Panchenkov, hemoglobin content - using the gemometer Sali (3, 4), phagocytosis - by the method of Cost and Stenko (5), the bactericidal activity of serum blood - by the method of O.V. Smirnova and T.A. Kuzmina (6), lysozyme activity - by the method of P.A. Emelyanenko (7).
Kidney function was assessed from the physicochemical characteristics of urine (color, clarity and consistence - visually determined), protein, glucose, ketone bodies, hemoglobin, pH - using the Penta PHAH test-strips («Brno», Czech Republic), the content of bilirubin and urobilin - using the Ikto PHAH test-strips («Brno», Czech Republic).
The experiment was also carried out on goslings of the Italian White breed (two groups of 300 birds in each group) in the poultry farm "Sputnik" (Orenburg reg.). The control group I: during the periods of 1-20 th, 21-65 th and 66-180th days of life, the goslings were fed the mixed fodder PK-30, PK-31 and PK-32. The experimental group II: the inactivated probiotic laktoamilovorin (made of L. amylovorus the strain BT-24/88) was added to the basic diet at a dose of 7 g per 100 kg of feed during the 1 st month of life. During the first 3 days of life, the inactivated preparation was also added to drinking water (0.7 g / l) at its unlimited consumption by goslings. Laktoamilovorin live concentrate containing 4,26 x 1010 viable cells/g was inactivated by boiling, dried at 120°С to constant weight, mixed with feed and dissolved in water immediately before feeding. During the experiment, all planned veterinary and technological activities were performed.
Blood samples for biochemical analysis were taken from the alary vein in five birds (three males and two females from each group) before the morning feeding. Bactericidal activity of blood serum was determined by the method of O.V. Smirnova and T.A. Kuzmina (6), the concentration of lysozyme - by Z.V. Ermol’eva and I.S. Buyanovskaya, the activity of b-lysine - by an accelerated method of O.V. Bukharin (8). The quality and chemical composition of geese meat were determined by conventional methods (9, 10).
Statistical processing of data was performed by standard methods of V.S. Asatiani.
Results. Feeding to rabbits of live and dead cultures of L. amylovorus BT-24/88 didn’t cause a significant impact on the number of red blood cells, ESR and hemoglobin content in the blood, while the number of leukocytes was significantly increased (P <0,01-0,001) (Table 1). WBC: the content of basophils, eosinophils, young and stab pseudo was similar in all groups, whereas the proportion of lymphocytes increased from 54-59% in control to 62-68% in rabbits fed the both live and dead cultures of Lactobacillus, owing to the reduced percentage of segmented pseudo.
Live and dead culture of L. amylovorus stimulated  humoral immunity. Adding to diet of live culture increased phagocytic, bactericide and lysozyme activity of blood serum (P <0,05-0,001) (see Table. 1). In earlier experiments of N.N. Maltseva et al. (2) on mice, was shown that the oral introduction of Enterococci or Bifidobacteria stimulated the increase in number of immunoglobulin-synthesizing cells in the lamina propria of small intestine. Feeding of dead Bifido-, Lactobacillus and Enterococcus promoted the substantial increase of survival rate of mice infected with Salmonella dublin C-96, whereas feeding of heat-inactivated bacteroids didn’t have a positive impact.

After the microbes’ penetration into the body, they are being absorbed and digested by tissue macrophages; the released antigenic peptides initiate cellular and humoral response by T-and B-cells. At the same time, macrophages excrete cytokines, which activate the factors of non-specific resistance (11).The immunomodulatory preparations of microbial origin contain the peptidoglycan components of bacterial cell wall as an active agent. The main target of its action - cells of the monocyte-macrophage level. It enhances almost all of their functions, increases the resistance to pathogenic bacteria, viruses, fungi, stimulates leucopoiesis and repair processes (12).
The foregoing data let us conclude that L. amylovorus the strain BT-24/88 as a component of the preparation laktoamilovorin has the pronounced immunomodulatory properties due to bacterial peptidoglycans in its composition.
Urine of rabbits of all groups had the straw-yellow color, specific smell, pH 7,0-9,0, no admixture of blood, hemoglobin, protein, sugar, nitrates and ketone bodies; though, a small amount of bilirubin was found, perhaps due to the retention of bile pigments to values exceeding their  kidney threshold (> 2 mg/100 ml).
Feeding of live and dead cultures of L. amylovorus caused a positive impact on livestock parameters. Adding to the diet of nutrient medium only promoted the increase in weight gain by 16,8%, feeding of live and dead culture of lactobacilli - by 41,6 and 34,0%, respectively (P <0,05). In the experimental group V, the weight gain wasn’t  significantly different from control (Table 2).
The deposition of external fat wasn’t significantly distinct between groups and varied within 3,9-5,5% from the weight of a carcass, internal fat retention in groups III, IV and V exceeded control in 1.75, 1.92 and 1.21-fold, respectively (see Table. 2).
Weight of internal organs in groups II and III did not differ significantly from those of control, though, in group IV the heart was lighter and the weight of thymus exceeded control by 44,3% (P <0,01); the least weight of the heart and liver was observed in group V.

At post mortem inspection of carcasses and internal organs of the rabbits of groups I, II and III, no visible pathological changes were found. Lymph nodes in the breast and abdominal cavities were not enlarged, the cut - grayish color, with no hemorrhages. Serous membranes - smooth and shiny. Lungs - elastic, trachea and bronchi - normal, with no swelling, abscesses and bleeding. The heart was not increased. Pericardium, epicardium and endocardium without infiltrates and hemorrhages. Parenchyma of the liver and spleen - cherry-colored, with no foci of necrosis and abscesses. Gall bladder - normal. In two rabbits from group IV, the liver of pale color was observed, and its weight exceeded the values in control. In group IV, one animal had the clay-colored liver with enlarged gall bladder, and another one - hemorrhages in the cortical layer of kidneys. In other animals of all experimental groups, kidneys were of normal size without inflammation and hemorrhages. Serous membranes of the stomach, small and large intestine - smooth and shiny. Mucous membranes - grayish-pink without hemorrhages and foci of inflammation.
Introduction to the diet of goslings of inactivated L. amylovorus the strain BT-24/88 did not harm their health. As in previous experiments with the active live preparation (13), the increase of survival rate was observed: in the control group, survival rate at 1-month- and 6 months-age was 90 and 86%, respectively, in the experimental group - 95 and 91%. Live weight of goslings fed the inactivated probiotics at the age of 30- and 120-days was 1496 ± 17 and 4382 ± 52 g and exceeded control by 5,9 and 4,5% (P <0,05); though, by the age of 6-months, this difference reduced to 3,4% in favor of the experimental group and was statistically insignificant.
Inactivated laktoamilovorin didn’t affect the dynamics of bactericidal activity of blood serum, concentration of lysozyme and the activity of  b-lysine, which were changing synchronously in both groups (Table 3). Inactivated feed probiotic induced non-specific resistance in young animals during the period of feeding the preparation (P <0,05), and during the 2nd month of life after its exclusion from the diet; later the differences leveled (Table 3). These data suggest that inactivated culture of L. amylovorus BT-24/88 stimulates non-specific resistance of poultry, apparently due to peptidoglycans, which are being released with the digestion of lactobacilli cell walls.
Improvement of the immune status of an organism induced a beneficial effect on birds’ productivity: the  live weight of 6-month-aged gooses of the experimental group was greater than in control by 3.36%, the weight of edible carcass parts - by 3,54% (P <0,05).

For all groups of gooses, there wasn’t found any significant differences in weight of muscles and bones, and in chemical composition of meat.
The results obtained in our experiments confirm the fact previously established by other authors: dead bacterial cultures can stimulate the immune system (2), which process is mediated by the immunomodulatory activity of peptidoglycanes of the bacterial cell wall (12).
So, adding to the diet of animals and poultry of live or inactivated culture of Lactobacillus amylovorus the strain BT-24/88 stimulates their non-specific resistance, survival rate and productivity.

REFERENCES

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7. Yemel’yanenko P.A., Seasonal Dynamics of Humoral Factors of Natural Resistance in Blood Serum of New Calves,  Dokl. VASNIIKhL, 1977, vol. 10, pp. 32-34.
8. Immunologicheskiye metody issledovanya v zhivotnovodstve / Pod red. R.P.Maslyanko (Immunological Methods of Investigation in Animal Husbandry / Ed. R.P. Maslyanko), L’vov, 1987.
9. Masliyeva O.V., Analiz kachestva kormov i produktov ptitsevodstva (Quality Tests of Fodders and Products in Aviculture), Moscow, 1970.
10. Bel’kov G.I., Kuranov Yu.F., Khrutskaya S.F. e.a., Otsenka myasnoj produktivnosti i kachestva myasa ybojnogo skota (Assesment of Meat Productivity and Quality of Meat in Slaughtered Cattle), Orenburg,1984.
11. Pinegin B.V.,  Current Views on Stimulation of Anti-Infective Immunity by Immunomodulatory Preparations, BIO, 2005,  no.1, pp. 2-4; no. 2, pp. 21-22. 12. Fedorov Yu.N., Immunocorrection: Application and Mechanism of Action of Immunomodulatory Preparations, Veterinariya, 2005, no. 2, pp. 3-6.
13. Tarakanov B.V., Gerasimenko V.V., and Nikulin V.N., The Metabolism and Productivity of Geese at  Adding to the Diet of the  Probiotic Laktoamilovorin,  S.-kh. biol, 2004, no. 4, pp. 52-58.

1Scientific Research Institute of Physiology, Biochemistry and Nutrition of Farm Animals, Russian Agricultural Academy, Borovsk 249013, Russia e-mail: bifip@kaluga.ru 2Orenburg State Agrarian University, Orenburg, 460795, Russia e-mail: probiotic2005@mail.ru

Received June 6, 2008

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