EFFECT OF BIFIDOBACTERIUM SP. ON THE HEALTH STATE OF PIGLETS, DETERMINED ON THE BASIS OF HEMATOLOGICAL AND BIOCHEMICAL INDICES

Małgorzata Kander

ABSTRACT

The studies were conducted on 70 piglets, divided into four groups: Group I - control – 10 piglets not receiving probiotics; Group II – 20 piglets given, starting from the 3^rd day after birth, live cells of Bifidobacterium breve and animalis; Group III – 20 piglets given, starting from the 3^rd day after birth, live cells of Bifidobacterium breve and animalis with an addition of bifidogenic substances; Group IV – 20 piglets given, starting from the 3^rd day after birth, a mixture of live cells of Lactobacillus acidophilus, Bifidobacterium breve and animalis with an addition of bifidogenic substances. All piglets aged 7, 14, 21, 28 and 35 days were subjected to clinical, hematological and biochemical examinations. Hematological examinations included the determination of: the count of red blood cells (Erys), the count of white blood cells (Lkcs), the hemoglobin level (Hb), the value of hemat

Key words: piglets, Bifidobacterium, probiotics, hematological and biochemical indices..

INTRODUCTION

In recent years much attention has been paid to probiotics, applied as therapeutic and preventive agents, and diet components in both humans and animals. Lactic acid bacteria (Lactobacillus, Bifidobacterium, Pediococcus, Leuconostoc, Enterococcus) and some strains of yeast (mainly Saccharomyces, Candida) and mould (Aspergillus oryzae, Aspergillus niger) are used most often in the production of probiotics of animal origin [24].

The main aim of using probiotic bacteria is to maintain the microbiological equilibrium in the alimentary tract, which can be achieved due to their properties, such as: good adhesion and colonization, survivability in the alimentary tract, competitiveness with pathogenic microflora, resistance to the low pH of gastric juices, the presence of bile acids and some metabolites produced during digestion, e.g. phenol [4, 5, 21, 30, 34, 39, 40, 51, 56]. Probiotic bacteria are also characterized by other positive properties, e.g. anticholesterol activity [2, 22, 23].

Probiotics may stimulate the immune system. The results of latest research show that rods of Bifidobacterium spp, Lactobacillus acidophilus and Lactobacillus casei stimulate in vitro the proliferation of B- and T-lymphocytes, cause an increase in leukocyte phagocytic activity and the level of interferon and immunoglobulin A [8, 18, 27]. Moreover, these bacteria are also characterized by antineoplastic properties, as they eliminate nitrates and nitrites, transformed in the alimentary tract in carcinogenic nitroso amines. They also limit the development of bacteria producing fecal procancerogenic enzymes, such as beta-glucuronidase or nitroreductase [6, 28, 50, 52].

Probiotics help to reduce and alleviate lactose intolerance. This indisposition, occurring in humans and young animals (piglets, calves, puppies), is caused by inactivity of beta-galactosidase, hydrolyzing milk sugar – lactose in the small intestine. Cells of probiotic bacteria release galactosidase in the alimentary tract, which contributes to lactose digestion and prevents intestinal disorders, such as diarrhea, constipation and distension with gas [26, 35].

Studies on Bifidobacterium were begun in 1900, when Tisser discovered a rod later called Bacillus bifidus communis during microscopic observations of the intestinal microflora in infants [3]. He also found that Bifidobacterium rods constitute autochtonic microflora of the alimentary tract, are among the first bacteria to colonize germ-free digestive tracts of newborns, and dominate in the large intestine microflora during lactation.

As regards their morphology, bifidobacteria are immobile, non-sporing and catalase-negative microorganisms. The optimum conditions for their development are a temperature of 37°C and pH 5.6 – 6.4. Most of them are anaerobic organisms, but particular species may differ in their sensitivity to oxygen [55, 58]. Some bifidobacteria are resistant to numerous antibiotics (gentamycin, neomycin, streptomycin, B polymyxin), which is important in antibiotic therapy and depends of their species and the amount of a given antibiotic. The growth of the majority of bifidobacteria is strongly inhibited by ampicillin, chloramphenicol, erythromycin, G penicillin and lincomycin [58].

Bifidobacterium rods ferment glucose producing acetic and lactic acids (L+). Apart from these acids, they can also produce small quantities of formic acid, ethanol, succinic acid, diacetyl, 2-pirolidon-5-carboxyl acid and bacteriocins [1, 33, 45].

The experiments conducted so far show that probiotic preparations have a beneficial effect on animals, manifested by higher body weight gains and better feed conversion [24, 48, 53], increased synthesis of vitamins B, better assimilation of Ca, P and Fe [25, 48], higher stress resistance, a quicker recovery [16, 48, 51] and a lower mortality rate among young animals [24, 25].

Prophylactic preparations are often given to piglets in the postnatal period. They contain mainly antibiotics, sulphonamides, vitamins and mineral compounds, and are usually applied in the form of feed supplements. However, they may have a negative influence of the health state of new-born piglets and result in antibiotic resistance of bacterial strains. That is why producers have recently started to administer probiotic preparations to piglets, as they are an alternative to antibiotics.

The aim of the present studies was to determine the effect of Bifidobacterium sp. on the health state of piglets. The effectiveness of the probiotics applied was examined on the basis of clinical observations, and hematological and biochemical analyses.

MATERIAL AND METHODS

The experimental material were 7 litters of piglets (Polish Landrace x Polish Large White x Złotnicka) of both sexes, staying with sows from the day of their birth and weaned at the age of six weeks. They were reared on large commercial farms, under standard sanitary conditions. The piglets were divided into four groups: Group I - control – 10 piglets not receiving probiotics; Group II - experimental – 20 piglets given, starting from the 3^rd day after birth, live cells of Bifidobacterium breve and animalis (10⁸ cells/cm³) <note 1>; Group III - experimental – 20 piglets given, starting from the 3^rd day after birth, live cells of Bifidobacterium breve and animalis with an addition of bifidogenic substances (peptides from enzymatic protein hydrolysis) <note 1>; Group IV - experimental – 20 piglets given, starting from the 3^rd day a fter birth, a mixture of live cells of Lactobacillus acidophilus, Bifidobacterium breve and animalis with an addition of bifidogenic substances (peptides from enzymatic protein hydrolysis) <note 1>.

All piglets aged 7, 14, 21, 28 and 35 days were subjected to clinical, hematological and biochemical examinations. Hematological examinations included the determination of: the count of red blood cells (Erys), the count of white blood cells (Lkcs), the hemoglobin level (Hb), the value of hematocrit (Ht), the mean hemoglobin concentration in the red blood cell (MCHC) and the mean volume of the red blood cell (MCV). Biochemical analyses of the blood serum comprised the determination of: the activity of lactate dehydrogenase (LDH), alkaline phosphatase (ALP), amylase (AM) and lipase (Lp); the levels of glucose (Glu), cholesterol (Chol), urea, total protein (TP), sodium (Na⁺), potassium (K⁺) and chloride anion; the parameters of the acid-base equilibrium (pH, pCO_2, HCO₃, BE).

The hematological analyses were made by commonly applied methods. The serum activity of amylase, LDH, ALP and lipase was determined by the kinetic method (Pointe Scentific tests). The serum level of total protein was determined by the end point method (Pointe Scentific), the levels of cholesterol and urea – by the enzymatic method, the level of glucose – by the oxidase method (Pointe Scentific). The biochemical indices were determined with a spectrophotometer Marcel s 330. The parameters of the acid-base equilibrium were determined with an analyzer Ciba –Corning, type 248. The electrolyte content (sodium - Na⁺, potassium - K⁺, chloride anion - Cl^-) was determined by the ion-selective electrode method, using an analyzer Easy Lyte. Blood samples were collected from vena cava cranialis, under identical conditions.

The results of laboratory analyses were presented in the SI units and analyzed statistically by the Duncan test. The results of hematological and biochemical examinations are presented in Tables and Figures.

RESULTS AND DISCUSSION

Clinical trials performed in the control group showed single cases of intestinal catarrh – its main symptom was diarrhea. The control piglets were characterized by slower development and considerable differences in body weights. The piglets from the experimental groups (II, III, IV) did not manifest any disease symptoms. The differences in body weight gains were here much smaller than in the control group. They were characterized by vitality and good appetite. The positive effect of probiotic preparations, reflected by higher body weight gains and better feed conversion, as well as a lower incidence of diarrhea and mortality rate of piglets, has been confirmed by many experiments [24, 44, 49, 53].

Hematological examinations made in the control groups showed lower hemoglobin levels in piglets aged 7 and 14 days – 5.19 mmol/l and 5.52 mmol/l respectively (Table 1, Figure 1). In the experimental piglets the hemoglobin content was within the physiological norms [29, 36, 57]. The red blood cell count in all piglets was increasing with age, but remained below the reference values. It was higher in the experimental piglets than in the control ones, and varied from 3.88 x 10¹²/l on the 7^th day after birth to 4.75 x 10¹²/l on the 35^th day (Table 1, Figure 2). These results indicate the so called physiological anemia in the control group, caused by rapid growth and insufficient iron supply [29]. Probiotic bacteria reduce the reaction of the alimentary tract, which results in better iron salt absorption from the small intestine. They also produce vitamins B, affectin g positively blood-forming processes, which was also confirmed by the results obtained in the experimental groups (Table 1, Figures 1, 2). A beneficial influence of probiotics, manifested by a higher hemoglobin level in the piglets from group II, compared with the control group, was also confirmed by statistical analyses (Table 1a, Figure 1). Bomba et al. [7] also noted an increase in the erythrocyte count and hemoglobin level in gnotobiotic piglets receiving Lactobacillus sp., but as late as three weeks after their birth. Similar observations were also made by Miller et al. [38].

The hematocrit value and the leukocyte count were within the physiological norms in all piglets [29, 36]. An increase in the mean red blood cell volume (MCV), accompanied by a decrease in the mean hemoglobin concentration in the red blood cell (MCHC) was observed in the control piglets, i.e. MCV: 7 days after birth – 68.5 fl, 14 days – 78.1 fl, 21 days – 78.4 fl, 28 days – 74.6 fl, 35 days – 77.3 fl; MCHC: 7 days after birth – 18.71 mmol/l, 14 days – 16.69 mmol/l, 21 days – 16.77 mmol/l, 28 days – 17.01 mmol/l, 35 days – 16.75 mmol/l (Table 1, Figures 3, 4). Fluctuations in the indices may suggest mild hypotonic dehydration, resulting from diarrhea. Similar differences in the values of MCV and MCHC were also noted in the experimental groups. MCV reached the highest values in the piglets aged 7 days from group II (81.3 fl) and in those aged 35 days from groups III and IV (80.2 fl and 85.1 fl respectively). MCHC was lower (Table 1, Figures 3, 4). Similar values of the hematological indices were obtained by Falkowski et al. [19], who applied a probiotic with Lactobacillus sp. According to Sjaasted et al. [54], the application of probiotic preparations with normal iron supply causes intensification of erythropoiesis, probably as a consequence of hypersynthesis of erythropoietin, reflected by fluctuations in the levels of MCV and MCHC. Nolte [41] claims that probiotics have a beneficial influence on erythrocyte indices.

There was a close correlation between the results of hematological examinations, the level of electrolytes and the parameters of the acid-base equilibrium. A decrease the serum level of sodium was observed in both the control and experimental groups. The level of potassium remained within the physiological values [29, 36]. The chloride anion content was below normal in 21- and 28-day-old control piglets – it amounted to 81.3 mmol/l and 89.1 mmol/l respectively (Table 2). In the experimental piglets the chloride anion content was within the reference values, but in groups III and IV displayed a falling tendency with age. A decrease in the blood pH and a lower level of bicarbonates and alkali reserve, accompanied by compensatory growth of partial pressure of carbon dioxide (pCO₂) – slight in the control group and considerable in the experimental ones - were observed in all piglets (Table 3, Figures 5, 6 , 7, 8). In the piglets from group II aged 7, 14, 21, 28 and 35 days pCO₂ was 8.9 kPa, 6.9 kPa, 22.9 kPa, 7.0 kPa and 10.2 kPa respectively. In the piglets from groups III and IV its highest values were noted 35 days after birth – 9.7 kPa and 9.4 kPa respectively (Figure 6). Significant alkali deficiency occurred in the piglets from groups III and IV, aged 7 and 14 days, i.e. /-/5.9 mmol/l, /-/6.0 mmol/l, and /-/6.3 mmol/l, /-/6.1 mmol/l respectively (Figure 7). These results indicate mild metabolic acidosis, and may be connected with not fully developed efficiency of the alimentary tract of young piglets. In the control piglets it could be additionally caused by slight organism dehydration resulting from diarrhea, and in the experimental ones – by the production of acid metabolites (lactic acid, acetic acid and propionic acid) by probiotic bacteria. Prohaszka and Baron [47] found secretory hypofunction of parieta l cells in piglets aged 3-7 weeks, leading to hyposecretion of hydrochloric acid. During its production, in the presence of carbonic anhydrase, HCO^-₃ ions are formed in the mucous cells of the stomach. The blood leaving the stomach during hydrogen ion secretion contains an excess of bicarbonates, referred to as the so called “alkaline influx”. This is an important source of buffer alkalis for the organism. The mechanisms discussed develop fully at the age of 8-9 weeks, which may be the cause for metabolic acidosis in young piglets, resulting from certain instabilities in homeostasis [11, 12]. Depta et al. [15, 17] also observed compensated metabolic acidosis in piglets aged 6 to 21 days receiving Lactobacillus sp., manifested by the blood pH shift towards acidity, a decrease in the level of bicarbonates and alkali reserve, accompanied by compensatory growth of CO₂ partial pressure.

The activity of the enzymes analyzed was within the physiological norms, both in the control and experimental groups, showing only slight fluctuations (Table 2). A statistically significant increase was noted in the amylase activity in the piglets from group III aged 7, 14, 28 and 35 days: 1536 IU/L, 1508 IU/L, 2047 IU/L and 1629 IU/L respectively (Figure 9). This may be connected with the intensity of digestion processes taking place in growing piglets. According to Corring et al. (9), the amylase activity is relatively low up to four weeks after birth (i.e. weaning), and then starts to grow gradually. This was also confirmed by own investigations.

Enhanced, though remaining within the physiological norms, activity of LDH was observed in all piglets as they were growing older (Table 2). Its highest values were found in 35-day-old piglets: group I - 431 IU/L, group II – 624 IU/L, group III 469 IU/L, group IV – 356 IU/L. This could be related to the so called “weaning stress”, often accompanied by significantly higher activity of liver enzymes [20]. Some authors claim that this is the right time for the administration of probiotics, as they can alleviate the effects of various kinds of stress [43].

Over the experimental period the activity of lipase displayed a falling tendency in all groups. This tendency was becoming stronger with age (Table 2). However, it should be emphasized that its values were lower in the experimental piglets (group II: 7 days after birth – 92 IU/L, 14 days – 26 IU/L, 21 days – 40 IU/L, 28 days – 21 IU/L, 35 days – 24 IU/L; group III: 7 days after birth – 66 IU/L, 14 days – 39 IU/L, 21 days – 29 IU/L, 28 days – 21 IU/L, 35 days – 34 IU/L; group IV: 7 days after birth – 52 IU/L, 14 days – 25 IU/L, 21 days – 27 IU/L, 28 days – 23 IU/L, 35 days – 19 IU/L) than in the control ones. Similar results were obtained by Depta et al. (13). They also noted a decrease in the lipase activity in the course of the experiment, but its values were somewhat lower in the control group than in the experimental one.

A decrease in the activity of alkaline phosphatase was observed in both the control and experimental groups (Tables 2, 2a, 2b, Figure 10). Its level was statistically significant in the piglets from group IV, reaching the lowest value on the 28th and 35th day after birth - 162 IU/L and 159 IU/L respectively. High activity of this enzyme in piglets on the first days after birth may be connected with intensive processes of bone formation and an increase in the osseous fraction of alkaline phosphatase. Later on, when the growth of piglets becomes less intensive, a decrease in the activity of ALP may be a consequence of a lower content of this fraction, and not fully developed mechanisms of intestinal digestion (low activity of the intestinal fraction).

The total protein level in all groups (I, II, III and IV) was within the reference values (Table 2, 2a, 2b). Karput and Pudenko [31], who applied probiotic preparations in piglets for the first five days after birth, noted considerably higher levels of total protein and immunoglobulins, whereas in the studies conducted by Depta et al. [13, 15] and Falkowski et al. [19] the level of total protein did not differ from normal.

The level of glucose demonstrated a falling tendency in all groups as the animals were growing older, but did not exceed the physiological norms [29, 36]. A highly significant decrease in glucose concentration was observed in the control piglets (7 days after birth – 6.16 mmol/l, 14 days – 5.27 mmol/l, 21 days – 5.60 mmol/l, 28 days – 5.59 mmol/l, 35 days – 5.72 mmol/l), suffering from diarrhea (Tables 2, 2a, 2b, Figure 11). A low glucose content in these animals could be caused by maldigestion and intestinal malabsorption. Keljo and McLead [32] found two glucose carriers in piglets, differing in the degree of affinity (high and low). According to these authors, animals with intestinal catarrh have only low-affinity carriers. Lack of high-affinity ones is an important factor of glucose malabsorption. In own research we noted a statistically significant increase in the glucose level in the experimental piglets. Its highest values were found in the piglets from group III, aged 7, 14, 21, 28 and 35 days. They were as follows: 8.41 mmo/l, 7.48 mmol/l, 7.97 mmol/l, 7.28 mmol/l and 6. 44 mmol/l respectively (Tables 2, 2a, 2b, Figure 11). This suggests that probiotics could positively affect glucose absorption from the alimentary tract. Huska and Bires (27), Depta et al. [15] and Falkowski et al. [19] believe that probiotics have no effect on the level of glucose. They did not observe an increase in its concentration in piglets. Its higher content was noted only in calves receiving Lactohydrat [16].

The level of cholesterol was characterized by a falling tendency in all groups. In 35-day-old piglets from group II its content (1.79 mmol/l) was much below normal [29, 36] (Tables 2, 2a, 2b, Figure 12). One of the properties of probiotic preparations is their anticholesterol activity. The serum level of cholesterol decreases as probiotic bacteria are able to degrade and assimilate this compound. Probably some metabolites of probiotic bacteria inhibit the estrification of cholesterol in intestinal mucosa, in this way reducing its level in the organism. Some cultures of intestinal bacteria reduce cholesterol to caprosterol, excreted with bile acid salts and their derivatives [50]. This anticholesterol activity was confirmed by numerous experiments on animals [23, 42, 43, 46]. Their authors obtained a statistically significant decrease in the levels of total cholesterol and the LDL fraction, but did not observe changes in the level of the HDL fraction.

According to Lettner and Preining [37], probiotic bacteria can reduce the level of ammonia in the alimentary tract and blood. This is possible due to the presence of lactic acid in the large intestine. This acid limits or inhibits harmful metabolism of proteins and amino acids, reducing the blood absorption of toxic substances (ammonia, amines, indole) produced as a result of their transformations. In own investigations the level of ammonia was within the physiological norms in the control group, and showed some fluctuations in the experimental ones. In 21- and 28-day-old piglets its level increased, reaching the following values: group II – 8.43 mmol/l and 7.28 mmol/l respectively, group III – 9.1 mmol/l and 8.2 mmol/l, group IV – 9.5 mmol/l and 11.2 mmol/l. In piglets aged 35 days it was back to normal (Tables 2, 2a, 2b, Figure 13). Depta et al. [15] noted a significant increase in the ammonia level in piglets receiving Lactobacillus sp., not changing with age. Bomba et al. [7] observed a considerable decrease in the ammonia level in gnotobiotic piglets given Lactobacillus casei, which confirms the reducing effect of probiotics.

The results of the clinical, hematologic and biochemical examinations show that Bifidobacterium sp., applied alone or in combinations with bifidogenic substances and Lactobacillus acidophilus, has no negative effect on piglets.

CONCLUSIONS

1. Both Bifidobacterium sp. rods and complex preparations (Bifidobacterium + Lactobacillus acidophilus + bifidogenic substances) have a positive influence on the health state of piglets. They normalize the levels of erythrocyte indices (preventing physiological anemia), cholesterol and glucose.

2. The probiotic preparations given to piglets caused mild acidification of their organisms (a tendency towards compensated metabolic acidosis), but did not disturb the acid-base equilibrium.

3. The application of pre- and probiotics to piglets on the first days after birth can help to prevent diarrhea and control stress.

Note 1 – the probiotic preparations were made at the Department of Industrial and Food Microbiology, Faculty of Food Sciences, University of Warmia and Mazury in Olsztyn.

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