Nowadays probiotics are widely used in animal nutrition, as evidenced by the variety of different types of bacterial feed additives on the EU market. Currently probiotics are used in feeding of poultry, pigs, sows, hens, turkeys, fattening cattle, dairy cows, lambs, sheep, goats, and rabbits (20). Probiotics are live microorganisms, mainly Gram-positive bacteria (
In order for a given probiotic strain to be marketed as feed additive, it must pass through the procedure for their authorization under Regulation (EC) No 1831/2003. Article 6 and Annex I of the Regulation provide categories and functional groups of feed additives. Probiotics are classified under the category “zootechnical additives”. In the case of registration of probiotic strains, the efficacy test of the additive must be performed on the target species. The positive effect of the probiotic supplement should be demonstrated by a decrease in the morbidity or mortality of the species tested and a more complete use of feed ingredients (20). Moreover, Commission Regulation (EC) No 429/2008 provides rules for the implementation of Regulation (EC) No 1831/2003 and imposes the obligation to provide a safety assessment (tolerance test) of the use of the probiotic additive by the target animals at the highest proposed levels in feed or water and at multiple levels.
The aim of a tolerance test is to provide a limited evaluation of the short-term toxicity of the additive to the target animals. It is also used to establish a margin of safety, if the additive is consumed at higher doses than recommended. Such tolerance tests must be conducted to provide evidence for the safety of the probiotic for the target animal. Test animals should be routinely monitored for visual evidence of clinical effects, performance characteristics, product quality, haematology and routine blood chemistry (7). Particular attention should also be paid to the acquisition of resistance to antibiotics and the transfer of resistance genes by probiotic strains used as feed additives (20).
The aim of the study was to provide an efficacy and safety assessment of the use of potentially probiotic lactic acid bacterium strains in the feed during broiler chickens rearing in the tolerance test.
The experiment lasted for 42 days. Chickens were reared in the production hall in the Agricultural Experimental Station Obory-Wilanów (in Warsaw) owned by Warsaw University of Life Sciences (SGGW). A total of 500 one-day-old Ross 308 chicks, initially weighing 44.4 ±0.58 g, were divided into four equal groups: three experimental groups: Max, 10Max, Min, and one control group K (in five repetitions). The birds were kept on straw litter in 20 pens (25 birds in each pen at a stocking density of 11.4 birds per m2). Air temperature, relative humidity and cooling conditions were the same for all the treatment groups. On the first day of life the chickens were vaccinated against the following diseases: Marek’s disease and Gumboro disease with Vaxxitek IBD vaccine (Merial, Germany) by injection; infectious bronchitis /IB/ with Cevac Bron 120 L vaccine, and Newcastle disease /ND/ with Cevac Vitapest L (Ceva Animal Health, France) vaccine by the spray method. On the 15th day of life the birds were vaccinated against infectious bronchitis /IB/ with Nobilis 4/91 vaccine (Intervet, Poland).
The broilers were fed according to the following feeding programme: starter 1–21 days, grower 21–35 days, and finisher 36–42 days. Feed and water were provided
The experimental design was as follows:
K: control group (without addition of preparation to feed);
Max: fed the maximum dose of the preparation as recommended by the producer;
Min: fed the minimum dose of the preparation as recommended by the producer;
10Max: fed a dose of the preparation ten times higher than the maximum recommended dose (tolerance group).
The number of live bacteria in the preparation was 1.0 × 109 cfu g–1. The quantities of active agents in the preparation (live lactic acid bacteria) introduced to the diets and the expected daily intake of bacteria (calculated on the basis of predetermined feed intake by birds) are presented in Table 1.
Calculated quantities of preparation and bacteria in feed and suspected daily intake of the preparation and bacteria by a chicken
The amount in feed
Daily intake
Experimental group
Preparation (g kg–1)
Bacteria (cfu g–1)
Preparation (g piece–1 day–1)
Bacteria (cfu piece–1 day–1)
Starter
Max
1.0
1.0 × 106
0.035
3.5 × 107
10Max
10
1.0 × 107
0.350
3.5 × 108
Min
0.5
5.0 × 105
0.018
1.8 × 107
Grower
Max
0.3
3.0 × 105
0.036
3.6 × 107
10Max
3.0
3.0 × 106
0.360
3.6 × 108
Min
0.15
1.5 × 105
0.018
1.8 × 107
Finisher
Max
0.2
2.0 × 105
0.043
4.3 × 107
10Max
2.0
2.0 × 106
0.430
4.3 × 108
Min
0.1
1.0 × 105
0.021
2.2 × 107
All broilers were individually weighed at the start of the experiment and then at the age of 21, 35, and 42 days. After 21, 35, and 42 days of rearing, the total feed consumption of starter, grower, and finisher diets was determined for each group. Based on these data, the mean live body weight of chickens, feed consumption, and feed conversion after all rearing periods were calculated. Mortality was monitored during the whole rearing period.
At the end of rearing (42 day) 12 broilers (6 males and 6 females) were taken from each group for slaughter. Blood was sampled from the wing vein for haematology and routine blood chemistry. A whole small intestine was collected for microbial analysis.
After slaughtering, the whole content of the small intestine was collected, weighed, diluted 10 times with saline, and then again diluted from 10–1 to 10–5. From the last three dilutions 0.1 mL was plated onto the appropriate medium for enumeration of intestinal microbiota (in triplicate).
Qualitative and quantitative microbiological studies included the following indicators:
total number of
The presence of bacteria from the
Blood samples (3 mL) were taken into EDTA polystyrene tubes for morphological testing. All blood samples were collected at the same time to minimise any changes in blood composition caused by circadian rhythm. Samples were stored in containers with ice to avoid protein denaturation and were delivered to a specialised veterinary diagnostic laboratory within two hours where blood smear examinations and morphological analyses were performed.
Samples of collected blood for biochemical analysis were centrifuged at 20°C (3,000 rpm) for 10 min. Plasma was stored at –20°C until analysis which was performed with the use of the following commercial tests: aspartate aminotransferase (AST) (kinetic method, cat. no. A7560), creatine kinase (CK) (kinetic method, cat. no. C7512), total protein (burette reaction, cat. no. T7528), uric acid (cat. no. U7580), and bile acids (enzymatic method, cat. no. DZ042A-K) (Pointe Scientific, USA).
Statistical analysis consisted of calculating the mean values and standard deviations of the tested parameters. The significance of differences in mean values was determined using the ANOVA test and post-hoc Tukey test or ANOVA rank Kruskal- Wallis test and multiple comparisons (in the case when the assumptions of the parametric analysis was not fulfilled,
Compared to the control group, bacteria addition to the feed increased the live body weight of chickens after the first period of rearing (at 21 days). After 21 days of rearing the average body weight of chickens in the use-level (Max and Min) and tolerance groups (10Max) was significantly higher than in the control group (K) (Table 2).
Effects of experimental feeding on body weight and body weight gain of broiler chickens (g) mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 mean values (in rows) marked witd various letters differ at significance level α = 0.05 SD – standard deviation
K
Max
10Max
Min
Rearing period
Mean value
SD
Mean value
SD
Mean value
SD
Mean value
SD
Body weight
1st day
44.9
3.39
44.7
3.25
43.6
2.57
44.6
2.74
21st day
698.9
90.41
739.6
99.62
737.0
99.08
765.2
110.15
35td day
1785.6
213.36
1811.0
251.62
1841.9
234.33
1910.9
233.42
42nd day
2428.8
272.71
2450.6
320.53
2563.1
314.74
2574.6
315.33
Body weight gain
1st – 21st day
656.7
22.79
696.3
44.30
695.3
33.25
727.3
7.97
22nd – 35td day
1083.8
17.27
1078.9
81.72
1105.3
57.86
1139.0
34.23
36td – 42nd day
646.6
27.88
636.0
32.64
719.9
65.4
673.1
79.64
1st – 42nd day
2387.1
21.12
2481.6
77.03
2450.2
153.5
2539.4
68.43
The final live body weight of chickens (after 42 days of rearing) in the 10Max and Min groups was significantly higher than in the control group (K), but there was no significant difference between the final body weight of chickens of the use-level (Max) and control (K) groups. There was also no significant difference between the final average live body weight of chickens from the tolerance (10Max) and use-level (Min) groups.
Body weight gain between all examined groups during the second (22–35 days) and the final (36–42 days) rearing periods did not differ significantly, and variation between body weight gains was confined to the first feeding period. After 21 days of rearing body weight gain was the highest in the Min group and significantly higher than in the control group. Body weight gain in the Max and 10Max groups was slightly but not significantly higher than in the control group.
After 42 days of rearing it was observed that the total body weight gain was the highest in the Min group but the body weight gain in this group did not differ significantly from the rest of the groups (Table 2).
Mortality among chickens from experimental groups fed bacterial preparation was lower than in the control group. Mortality among chickens from the control group was the highest at 3.2%. Among chickens from the tolerance group (10Max) this parameter was no different from its value in the Max group, and lower than it was in the Min group (Table 3).
Effects of experimental feeding on mortality of broiler chickens (%) Chickens with very low body weight
Group
K
Max
10Max
Min
Mortality
3.2
1.6
1.6
2.4
1.6
0.8
0.8
0.0
Total losses
4.8
2.4
2.4
2.4
Generally mortality was low so no veterinary interventions were required. The reasons for losses (stated by the veterinarian) were: gout, colibacillosis, anaerobes enteritis and inflammation of the gall bladder.
Total feed consumption during the 42 days of the growing period exceeded 4.5 kg per chicken. There were no significant differences between total feed intakes among the experimental groups fed bacterial preparation. A difference was observed in the case of total feed intake per chicken between the control (K) and Max groups (chickens from the control group consumed less feed). In all groups chickens ate a similar amount of starter, grower, and finisher feed (Table 4).
Effects of experimental feeding on feed intake and feed conversion by broiler chickens mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 mean values (in rows) marked with various letters differ at significance level α = 0.05 SD – standard deviation
Feed
K
Max
10Max
Min
Mean value
SD
Mean value
SD
Mean value
SD
Mean value
SD
Feed initake, kg per chicken
Starter
1.36
0.06
1.46
0.06
1.4
0.08
1.47
0.09
Grower
2.15
0.22
2.21
0.15
2.06
0.2
2.08
0.13
Finisher
1.08
0.1
1.1
0.12
1.04
0.14
1.09
0.13
Total 1st – 42nd day
4.28
0.22
4.88
0.31
4.45
0.27
4.54
0.23
Feed conversioin, kg kg-1 of body weight gain
Starter
1.96
0.2
2.48
0.3
2.09
0.11
2.08
0.13
Grower
1.98
0.2
2.06
0.23
1.83
0.2
1.81
0.12
Finisher
1.67
0.14
1.71
0.13
1.51
0.14
1.66
0.29
Total 1st –M2nd day
1.89
0.13
1.97
0.1
1.81
0.13
1.84
0.08
In the case of feed conversion only one difference was observed, and it concerned starter feed conversion between the control and Max groups (conversion of starter feed in the control group was significantly lower than in the Max group) (Table 4).
In the case of females differences between experimental groups were observed in AST, total protein, and bile acid level. In all groups receiving bacteria bile acid levels were slightly higher than that of the control group. In the 10Max group the activity of AST was the highest. However, differentiation in particular biochemical parameters among groups receiving bacteria was not so clear. In the case of males no differences in biochemical parameters were observed between any experimental groups (Table 5).
Effects of experimental feeding on biochemical parameters of blood serum of broiler chickens mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 mean values for hens (in columns) marked with various letters differ at significance level α = 0.05 ± standard deviation
Group
Sex
AST (U L–1)
Total protein (G L–1)
CK (U L–1)
Bile acids (μmol L–1)
Uric acid (Mg dL–1)
K
♂
379.3 ± 61.82
55.6 ± 3.27
9187.5 ± 1704.24
11.0 ± 6.14
2.5 ± 0.93
♀
406.1 ± 47.03
27.0 ± 1.41
9481.4 ± 2086.87
6.96 ± 4.93
2.9 ± 1.12
Max
♂
451.0 ± 139.77
26.8 ± 2.71
10586.9 ± 1090.91
18.4 ± 2.98
7.7 ± 1.30
♀
375.1 ± 95.7
29.8 ± 2.56
9272.8 ± 1489.16
14.9 ± 24.79
4.7 ± 2.13
10Max
♂
405.4 ± 150.41
24.8 ± 2.79
9062.0 ± 2338.61
17.05 ± 4.62
4.0 ± 0.72
♀
563.5 ± 104.67
26.0 ± 1.67
11325.0 ± 602.84
13.67 ± 2.74
4.4 ± 1.03
Min
♂
417.5 ± 8.55
25.0 ± 1.73
10355.9 ± 887.5
10.1 ± 2.9
2.1 ± 0.21
♀
417.1 ± 91.2
28.0 ± 2.58
9953.6 ± 1654.55
15.25 ± 3.87
3.0 ± 0.95
In the case of haematological parameters of male and female chickens, no significant differences were observed among all experimental groups, P > 0.05. There was also no regularity related to the effect of the bacteria added to the diets on the analysed blood parameters. The biggest differences, however statistically insignificant, were observed among female 10Max and Min groups in the case of leukocyte count, which was much lower in these groups compared to the control group. The opposite situation was observed for male 10Max and Min groups, in these groups the content of leukocytes was higher, but still statistically insignificant, compared to the control group. Erythrocyte, lymphocyte, and haemoglobin contents in experimental groups fed bacteria was lower in male groups and higher in female groups compared to the controls, regardless of the dose of bacteria (Table 6).
Effects of experimental feeding on haematology of broiler chickens ± standard deviation
Group
Sex
Erythrocyte Total RBC (T L–1)
Lymphocyte (%)
Monocyte (%)
Leukocyte Total WBC (G L–1)
Haemoglobin (g dL–1)
Haematocrit PCV (%)
Heterophil (%)
Basophil (%)
K
♂
3.0±0.53
22.0±9.17
8.3±3.25
23.3±11.18
13.7±2.25
33.5±5.6
65.3±7.77
4.7±2.31
♀
2.5±0.16
43.0±0.89
4.3±1.91
41.7±5.92
11.7± 1.73
27.4±6.56
51.0±1.0
3.7±1.53
Max
♂
2.6±0.1
20.3±0.58
9.0±5.57
15.4±1.31
12.5±0.53
29.1±2.49
59.7±5.69
9.7±0.58
♀
2.6±0.2
38.7±14.57
7.7±6.51
42.3±26.15
12.2±0.75
25.0±4.36
49.0±9.54
5.7±2.08
10Max
♂
2.4±0.14
35.3±7.02
9.0±7.55
30.9±21.92
11.8±0.6
27.6±4.36
53.3±1.15
2.3±0.58
♀
2.8±0.28
39.0±7.0
2.3±1.15
19.1±6.1
13.3± 1.56
30.9±3.21
51.7±6.51
7.0±3.61
Min
♂
2.8±0.42
22.7±7.09
13.0±2.65
25.7±5.49
13.3±2.26
33.9±7.23
58.7±8.33
11.7±2.08
♀
2.7±0.04
39.3±14.47
14.3±2.08
24.3±5.41
12.6±0.46
29.1±1.44
51.3±11.5
3.3±1.53
After 42 days of rearing, microbiological analysis of intestinal contents showed the positive effect of LAB feeding on intestinal microbiota composition. In the intestinal content of chickens from all groups fed bacterial preparation,
The effects of experimental feeding on microbial analysis of intestinal content after 42 days of chickens rearing (cfu g–1)
Group
K
1.8 × 107
2.2 × 104
3.8 × 106
present
1.0 × 107
4.0 × 108
Max
5.2 × 107
not detected
7.3 × 106
not detected
1.3 × 107
2.1 × 108
10Max
1.0 × 107
not detected
2.3 × 106
not detected
4.6 × 107
8.4 × 108
Min
1.0 × 107
not detected
7.0 × 106
not detected
2.7 × 107
4.3 × 108
A wide variety of bacterial species are used as probiotics in animal nutrition. Some of them are little known and their use may represent a risk for the target species. The European Food Safety Authority (EFSA) has proposed a system for a pre-market safety assessment of selected groups of microorganisms. Microorganisms which raise safety concerns could be granted QPS status (Qualified Presumption of Safety) and be released from a full safety assessment (toxins or virulence factors should be demonstrated to be absent or of no concern) (21).
According to published data the effect of probiotics on the performance of chickens can be various. No positive effects were reported of probiotic
A beneficial effect of probiotics on broiler chicken body weight is not imparted in every instance and depends on the strain used or their particular combination. In this study the impact of the
The impact of probiotics on feed consumption can also vary depending on the strain used. The feeding of broiler chickens with probiotics resulted in a significant reduction in feed conversion ratio (FCR) and feed consumption per kg of bird weight gain compared to birds not fed probiotics (16). Meanwhile no effect of probiotic addition to feed (
An important factor influencing the effectiveness of probiotics in poultry is also the manner and time of their administration. It has been shown that administration of probiotics in feed, compared to drinking water, contributed to a higher mean daily body weight gain (24).
Blood examination is performed as a screening procedure to assess general health. Clinical signs of illness in birds are frequently subtle, so clinical chemistry is necessary to evaluate cellular changes (23). However, it is difficult to compare the results of blood or biochemical parameters of birds with other authors’ results because of the influence upon them of diet, age, rearing behaviour, environmental conditions, bird species, and sex (2, 12). On the other hand, according to Mazurkiewicz (15), the reference values of blood biochemical parameters for broiler chickens are the same for both sexes.
A key tool in the diagnosis of certain diseases is the determination of enzyme activity in the metabolic profile. Increasing enzyme activity in blood may indicate damage to cellular structures and is proportional to its degree (13). Interpretation of the assay results, however, is difficult due to the wide range of enzyme activities (9).
AST activity is currently considered to be a very sensitive but nonspecific biomarker of hepatocellular disease and is used with the muscle-specific enzyme creatine kinase (CK) to differentiate between liver and muscle damage (10). In the presented study CK was slightly above the reference value in all experimental groups, but AST was at a very high level (384–563.5 U L–1), much higher than the maximum reference value (15). AST and CK activities increase under pressure (
Total protein is important information used to establish supportive care. A decline in total protein may indicate protein-losing nephropathy, enteropathy or liver failure. In the case of total protein insufficiency, increasing dehydration should be investigated (10). Total protein in all experimental groups in this study was consistent with the reference values reported in the literature (6, 15).
Uric acid is a product of the catabolic breakdown of proteins and its concentration in serum depends on age, diet, and reproduction (23). According to Harr (10), increasing concentration of uric acid is due to renal disease and decreasing concentration is caused by liver failure. The concentration of uric acid in all experimental groups in this study was in accordance with the references values (6, 15), so no damage to said organs was observed. Recorded by Albokhadaim
In this study no significant escalatory effect of LAB administration on bile acids in chickens’ blood was observed. Bile acids are used to assess liver function. A concentration of bile acids higher than 75 μmol L1 suggests hepatic insufficiency, while a concentration higher than 100 μmol L–1 is diagnostic for decreased liver function, which could be caused by exposure to environmental hazards such as feed contamination (10).
In literature amassed data on haematological values of broiler chickens’ blood are limited. Haemoglobin, PCV and eosinophils of male chickens were higher than appropriate parameters of females but the percentage of lymphocytes in females was higher than in male chickens (23). Values for PCV, WBC, heterophils, lymphocytes, and basophils of the males differed from those of the females. Moreover, biochemical parameters, such as ALT, AST, and total protein were also different depending on sex (1).
Even less information is available about the impact of probiotics on haematological parameters of broiler chickens’ blood. Male broilers were fed probiotic (
Many studies have so far confirmed the ability of
In another study the probiotics (
In conclusion, the positive effect of the addition to diets of a bacterial preparation consisting of the two potentially probiotic lactic acid bacteria strains:
It can also be assumed that feeding broiler chickens with LAB addition influences mortality by reducing the number of deaths caused by various diseases. Moreover, feeding chickens with a bacterial preparation has a positive impact on the intestinal microbiota, due to inhibition of the growth of
The safety of the proposed bacterial feed additive was attested to by the failure of a dose of bacterial additive ten times higher than the maximum recommended to have any greater influence on examined health indicators, such as biochemical and haematological parameters of blood, than the maximum recommended dose.