1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Effect of Bacillus subtilis PB6 (CLOSTATTM) on performance of laying hens.
Evelyne Delezie1*; Liesbet Thijs2; Luc Maertens1; S. Decampeneere1
1
ILVO Animal Sciences Unit, Melle, Belgium, 2Kemin Europa N.V., Agri-Foods Division, Herentals, Belgium
*Corresponding author. Email: evelyne.delezie@ilvo.vlaanderen.be
Abstract
The objective of this trial was to determine the efficacy of Bacillus subtilis PB6 (strain
Bacillus subtilis ATCC PTA-6737) on laying hens’ performance. The strain was provided as a
premixture and named CLOSTATTM Dry. The zootechnical trial consisted of 2 dietary
treatments with 8 replicates of 18 laying hens each. The dietary treatments consisted of a
control diet (TrA) and the control diet supplemented with Bacillus subtilis PB6 at 1x108
CFU/kg feed (TrB). Laying hens (Lohmann brown), 21 weeks of age at the start of the
experiment, were used in this trial. Laying hens given the Bacillus subtilis PB6 performed
significantly better than the control group, resulting in a significantly higher laying rate
(P=0.007), egg weight (P=0.036), daily egg mass (P<0.0001) and better feed conversion
(P=0.005). The incidence of soft-shelled eggs and of dirty eggs was similar for all groups
whereas the incidence of cracked eggs was significantly lower (P<0.0001) when laying hens
were given the supplemented diet.
Key words: laying hens; probiotics; performance
Introduction
Bacillus subtilis PB6 (strain Bacillus subtilis ATCC PTA-6737) is a unique and natural
Bacillus subtilis strain and is a proprietary, spore forming, gram-positive bacterium. Bacillus
subtilis ATCC PTA-6737 is authorized for chickens for fattening in feed legislation by
Regulation (EC) 107/2010 under number 4b1823 in the category zootechnical additive and
functional group of gut flora stabilizers. An objective measurement of the efficacy of new
products is an essential step in the development of (cost) effective additives.
The objective of the current trial was to provide efficacy data of Bacillus subtilis PB6 for use
in laying hens. The strain is supplemented with carrier i.e. maltodextrin and limestone and
provided as a premixture, named CLOSTATTM Dry. For this purpose Bacillus subtilis PB6
was introduced in a basal diet. The bio efficacy of Bacillus subtilis PB6 for laying hens was
determined in a performance trial.
Material and Methods
Laying hens (Lohmann brown), 21 weeks of age at the start of the experiment, were used in
this trial. The birds were housed in the poultry experimental facility of ILVO/ Section Small
Stock Husbandry. The trial consisted of 2 treatments, each with 8 replicates of 18 animals.
Duration of the trial was 168 days as there were 6 periods of each 28 days. The trial was
designed as a 2-factorial Complete Block Design (2 treatments x 8 replicates = 16 pens) with
18 medium weight laying hens per pen with a total number of 288 birds. The test product,
provided as a premixture having spore counts of 2 x 108 CFU/g, was dosed at a rate of
500g/tonne, which resulted in a dosage of 1 x 108 CFU/kg feed. The different treatments were:
Treatment A = basal diet, Treatment B = basal diet + Bacillus subtilis PB6 (1 x 108 CFU/kg
feed).
Average pen body weight was recorded at the start and at the end of the experiment. Feed
intake was recorded and feed conversion and daily feed intake per bird were calculated for
each period. Daily number of eggs per pen unit were counted and every 2 weeks eggs were
1
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
weighed. Furthermore, every day the incidence of cracked, soft-shelled and dirty eggs was
recorded. Daily mortality and cullings were recorded for each pen. Egg quality was measured
in terms of egg shell deformation (under a 500 g load, in micrometer) at day 21 of each 28 dperiod. All parameters were subjected to a factorial analysis of variance (“2 x 8 x 6 / 2 dietary
treatments x 8 blocks x 6 period”) and the corresponding LSD-multiple range test (with P:0.05
as the threshold value).
Results and discussion
The main zootechnical results are presented in Table 1. There was no block (replicates) effect
but a significant effect of period on laying rate, egg weight, daily egg mass and feed
conversion was observed. Laying rate was significantly lower for the first period as hens were
only 21 weeks of age. Egg weight and daily egg mass significantly increased with the age of
the laying hens, therefore significantly higher egg weights and daily egg masses were reached
at the end of the trial. Daily feed intake of the laying hens ranged from 111.53 to 116.22 g/hen
with the highest feed intake during period 2 and 5. This resulted in a significant better feed
conversion during period 6 and the worst feed conversion ratio was obtained during period 1
caused by the significantly lower laying rate. Supplementing the feed with Bacillus subtilis
PB6 did affect the zootechnical parameters. Laying hens which received the supplemented
diet showed a significantly higher laying rate, egg weight and daily egg mass compared to the
control birds. Daily feed intake of both treatment groups did not differ significantly what
resulted in a significant better feed conversion ratio for the birds given the Bacillus subtilis
PB6 supplemented diet. The results of this trial are in line with results of previous in vivo
trials in chickens for fattening showing positive effects on performance. An improved weight
gain and feed efficiency of the chickens upon inclusion of Bacillus subtilis PB6 into the diet
has been shown (Kemin data not shown). Furthermore, microbiological evaluation of the
intestinal bacterial microflora of broilers has demonstrated that the counts of Clostridium
perfringens decreased with increasing dosage of Bacillus subtilis PB6 in the feed (Teo and
Tan 2004). Egg shell deformation was determined as a measure for egg quality.
Supplementing Bacillus subtilis PB6 (Tr B) did result in the significantly highest egg shell
deformation. There was also a significant effect of period with the lowest egg shell
deformation during period 3 and the highest during period 1.The number or % of dirty eggs
was very low and comparable between treatments. There was also no effect of dietary
treatment on percentage soft-shelled eggs. Percentage soft-shelled eggs was 0.52 and 0.47% if
laying hens received the control and supplemented diet, respectively. However, the incidence
of cracked eggs was significantly lower (P<0.0001) when laying hens were given the
supplemented diet (2.42 vs 1.34 %). At the start and at the end of the trial mean hen weight
was similar for all dietary treatments. Therefore, growth of the laying hens for both treatments
was not significantly different during the whole duration of the experiment. Also, mortality
percentage was not different between treatments and periods.
Conclusion
Positive effects of Bacillus subtilis PB6 on the overall performance parameters in this layer
study were present and did reach the significance level (P < 0.05).
References:
Teo A Y. H-M Tan. 2004. The effect of CloSTAT on pathogenic and beneficial bacteria in
broilers. Proceedings XXII World Poult. Congress, Istanbul, Turkey.p.174.
2
99
Table 1. The effect of dietary treatment and period on laying rate (%), egg weight (g), daily egg mass (g), daily feed intake (g/a) and
feed conversion
Daily feed intake,
Treatment
Laying rate, %
Egg weight, g
Daily egg mass, g
Feed conversion
g/a
Treatment
Control
90.84b
62.61b
57.06b
113.24
2.02b
Control +
93.19a
63.04a
58.93a
114.60
1.97a
CLOSTATTM
SEM
0.80
0.35
0.74
0.51
0.03
Period
ANOVA
Period
Treatment
Period x
Treatment
a-c
100
101
1
2
3
4
5
6
SEM
78.52b
96.13a
94.60a
96.00a
95.81a
94.58a
0.80
56.94d
62.46c
63.88bc
64.09b
64.51b
66.79a
0.35
44.73c
60.04b
60.44ab
61.53ab
61.81ab
63.16a
0.74
111.53
114.95
112.59
113.01
116.22
113.96
0.51
2.51b
1.91a
1.87a
1.84a
1.88a
1.81a
0.03
< 0.0001
0.0007
< 0.0001
0.036
< 0.0001
< 0.0001
NS
NS
< 0.0001
0.005
NS
NS
NS
NS
NS
means with the same letter are not significantly different from each other at P ≤ .05.