Full text of DOI:10.1093/japr/11.4.379

2002 Poultry Science Association, Inc.
Influence of Calsporin on
Commercial Leghorns
S. S. Sohail, M. M. Bryant, R. A. Voitle, and D. A. Roland, Sr.1
Department of Poultry Science and Alabama Agricultural Experiment Station,
Auburn University, Alabama 36849
Primary Audience: Nutritionists, Feed Manufacturers, Egg Producers
SUMMARY
A study was conducted to determine if Calsporin (Bacillus subtilis C-3102), a direct-fed microbial
(probiotic), improved performance of commercial Leghorns and eggshell quality. Hyline W36 hens
(n = 1,440; age = 65 wk) in 90 groups (16 hens/group) were randomly allocated to three dietary
protein levels (17.3, 16.1, and 15.0%), and three Calsporin levels (0.0, 0.003 and 0.006%) in a 3 ×
3 factorial for 10 wk. A Calsporin × protein interaction was observed on egg specific gravity (SG;
P < 0.01). Egg SG increased more in hens fed the higher Calsporin level than in hens fed the lower
level when dietary protein was 15%. This Calsporin level effect was reversed as dietary protein
was increased to 16.1%. Trends similar to that of SG were also observed on eggshell thickness.
Increasing dietary protein had a beneficial effect (P < 0.001) on egg production, feed consumption,
and egg weight. Addition of Calsporin to hen diets had no significant influence on feed consumption,
egg production, egg weight, or body weight of hens. More research is required to optimize any
potential benefit of Calsporin with respect to dietary protein on eggshell quality.
Key words: Calsporin, eggshell quality, Leghorns, probiotic
2002 J. Appl. Poult. Res. 11:379–387
ronmental [13, 14] effects to improve eggshell
quality. Also, several dietary supplements have
DESCRIPTION OF PROBLEM
In spite of the thorough and detailed infor-
mation available on factors influencing egg-
shell quality [1, 2, 3, 4], egg producers are
losing millions of dollars annually due to losses
associated with poor shell quality [5]. Eggshell
quality has always challenged egg producers
and layer nutritionists because of its economic
importance [6] and its roles in containing egg
contents and retaining internal egg quality. A
decline in eggshell quality increases the num-
ber of undergrades causing losses to the egg
processors and producers. Previous research
has focused on physiological [7, 8, 9], nutri-
tional [3, 10, 11], mechanical [12], and envi-
been tested in commercial layers to improve
shell quality [15, 16]. Trials have also been
conducted to study the internal environment of
the gut and determine mechanisms involved in
the absorption of nutrients in maintaining shell
quality [17, 18, 19].
Research has shown that under healthy and
nonstressful conditions, beneficial microflora
colonize gut surfaces in a symbiotic relation-
ship with the host and suppress undesirable
microbes that may be pathogenic [20]. Direct-
fed microbials, commonly known as probiot-
ics, have been found to establish a balance
between beneficial and pathogenic bacterial
1 To whom correspondence should be addressed: droland@acesag.auburn.edu.

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JAPR: Research Report
TABLE 1. Ingredient and nutrient composition of experimental diets
Diet
Ingredient
1
2
3
(%)
Corn (8.6% CP)
Soybean meal (48% CP)
LimestoneA
61.52
25.10
7.04
65.65
21.70
7.05
68.99
18.94
7.05
HardshellA
2.00
2.00
2.00
Poultry oil
1.59
0.87
0.29
Dicalcium phosphate
(Ca 21.7%; P 18.5%)
NaCl
1.63
0.46
0.25
0.25
0.17
0
1.64
0.46
0.25
0.25
0.13
0
1.66
0.46
0.25
0.25
0.11
0
Vitamin premixB
Mineral premixC
DL-Methionine
CalsporinD
Calculated analysis
Crude proteinE (%)
ME (kcal/kg)
Met + Cys (%)
Lys (%)
17.34
2,823
16.06
2,823
15.02
2,823
0.74
0.92
4.00
0.61
0.40
0.67
0.83
4.00
0.60
0.40
0.62
0.75
4.00
0.59
0.40
Ca (%)E
Total P, (%)E
Nonphytate PE (%)
ACalcium carbonate was supplied as limestone (16 × 120 US mesh, 1.19 to 0.125 mm) and hardshell (4 × 8 US mesh, 4.75
to 2.36 mm).
BProvided per kilogram of diet: vitamin A (as retinyl acetate), 8,000 IU; cholecalciferol, 2,200 IU; vitamin E (as dl-α
tocopheryl acetate), 8 IU; vitamin B12, 0.02 mg; riboflavin, 5.5 mg; D-calcium pantothenic acid, 13 mg; niacin, 36 mg;
choline, 500 mg; folic acid, 0.5 mg; thiamin mononitrate, 1 mg; pyridoxine, 2.2 mg; d-biotin, 0.05 mg; menadione sodium
bisulfite complex, 2 mg.
CProvided per kilogram of diet: manganese, 65 mg; iodine, 1 mg; iron, 55 mg; copper, 6 mg; zinc, 55 mg; selenium, 0.3
mg.
DCalpis Co., Ltd., Kanagawa, Japan. Calsporin levels of 0.003 and 0.006% were supplemented to these diets to create the
nine treatments.
EDietary protein, Ca, total P, and nonphytate P values were determined by chemical analysis.
populations in the intestine [20, 21, 22, 23].
Previous research has indicated beneficial ef-
fects of direct-fed microbials on egg produc-
tion (EP), feed conversion, and yolk choles-
terol [23]. Bacillus subtilis, a direct-fed micro-
bial that produces amylases and proteases, has
been shown to withstand high temperatures of
pelleting [22] and may prove beneficial to the
feed industry.
Calsporin, a commercially available Bacil-
lus subtilis-originated direct-fed microbial has
been reported to improve live performance and
microbiological status in broilers [20, 24]. Re-
search on the influence of Calsporin on the
performance of commercial layers is not yet
available. Induced molting is a popular man-
agement technique used in the commercial egg
industry [25] to extend the period of production
[26] and hen performance. Little research is
available on the nutrient requirement of molted
hens [27]. Therefore, the objective of this study
was to determine the influence of Calsporin on
performance and eggshell quality of commer-
cial Leghorns after induced molting.
MATERIALS AND METHODS
A 10-wk study was conducted on Hyline
W36 hens in their second cycle (molted hens)
to determine if Bacillus subtilis C-3102, com-
mercially available as Calsporin, has a benefi-
cial effect on hen performance, particularly
eggshell quality. This experiment used three
dietary protein levels (17.3, 16.1, and 15.0%)
and three Calsporin levels [0.0 (control), 0.003,
and 0.006%], and data were analyzed as a 3 ×
3 factorial. All nine dietary treatments were

SOHAIL ET AL: LAYER RESPONSE TO CALSPORIN
381
TABLE 2. Influence of Calsporin on egg specific gravity of Hy-Line W36 hens (second cycle, 69 to 79 wk of age)
Egg specific gravity (unit)
Week
Treatment
2
4
6
8
10
Mean
Protein (%)
17.3
16.1
15.0
SEM
1.0798b
1.0816a
1.0805b
0.0004
1.0818b
1.0826a
1.0825a
0.0002
1.0813b
1.0819ab
1.0822a
0.0003
1.0808a
1.0814a
1.0811a
0.0003
1.0801a
1.0805a
1.0805a
0.0002
1.0808b
1.0816a
1.0814a
0.0002
Calsporin (%)
0
1.0803
1.0809
1.0808
0.0004
1.0821
1.0825
1.0822
0.0002
1.0819
1.0816
1.0819
0.0003
1.0809
1.0812
1.0812
0.0003
1.0804
1.0805
1.0802
0.0002
1.0811
1.0813
1.0813
0.0002
0.003
0.006
SEM
Protein (%)
17.3
×
Calsporin (%)
0
1.0794a
1.0806a
1.0794a
1.0815a
1.0822a
1.0815a
1.0810a
1.0815a
1.0813a
1.0801a
1.0811a
1.0812a
1.0798a
1.0802a
1.0803a
1.0804a
1.0811a
1.0808a
0.003
0.006
16.1
15.0
0
1.0811a
1.0824a
1.0814a
1.0827ab
1.0832a
1.0818b
1.0822a
1.0818a
1.0816a
1.0814ab
1.0823a
1.0806b
1.0806ab
1.0811a
1.0797b
1.0806b
1.0822a
1.0810ab
0.003
0.006
0
1.0803a
1.0798a
1.0815a
0.0006
1.0820a
1.0822a
1.0833a
0.0004
1.0824a
1.0814a
1.0828a
0.0004
1.0812a
1.0803a
1.0818a
0.0005
1.0808a
1.0801a
1.0804a
0.0004
1.0813ab
1.0807b
1.0820a
0.0003
0.003
0.006
SEM
Significance
Protein
Calsporin
**
*
NS
*
*
NS
NS
NS
NS
**
NS
NS
NS
**
NS
**
NSa
NS
Protein × Calsporin
a,bMeans within a column and treatment section having different superscripts are significantly different (P < 0.05).
ANot significant at P > 0.05.
*P < 0.05.
**P < 0.01.
***P < 0.001.
isocaloric (Table 1). Experimental hens (n =
1,440) were divided into 90 replicates (groups)
of 16 hens each, such that hens from each repli-
cate had no access to feed or water of other
replicates. Each replicate had four adjacent
cages housing four birds per cage (31 × 41 cm).
A cage was left vacant between the replicates
to avoid cross-contamination through feed and
water. Replicates were also equally divided
between the two cage tiers.
Molting of hens was initiated at 65 wk of
age, and the experiment was run from 69 wk
of age until 79 wk of age. Prior to commence-
ment of the study, postmolt feed was supple-
mented for 2 wk with one of three Calsporin
levels to acclimate hens to Calsporin levels.
All 90 replicates were equally and randomly
assigned to the prestudy treatments and were
fed for 2 wk. To avoid contamination between
levels, diets with the same Calsporin level were
assigned a color-marked scoop for feeding the
hens. In addition, feeder troughs were also
marked with color (similar to scoops) to avoid
any accidental contamination among treat-
ments. At commencement of the study, 10 rep-
licates within each Calsporin level were ran-
domly allocated to each protein level (Table 1).
Feed and excreta samples were analyzed
periodically to monitor cross-contamination
between Calsporin levels, maintain an accurate

382
JAPR: Research Report
TABLE 3. Influence of Calsporin on eggshell thickness and body weight of Hy-Line W36 hens (second cycle, 69
to 79 wk of age)
Eggshell thicknessA
(mm)
Body weightB
(kg)
Treatment
Protein (%)
17.3
16.1
15.0
SEM
0.31
0.32
0.32
0.005
1.632
1.618
1.568
0.023
Calsporin (%)
0
0.31
0.32
0.31
0.005
1.586
1.627
1.605
0.023
0.003
0.006
SEM
Protein (%)
17.3
×
Calsporin (%)
0
0.31
0.32
0.31
1.536
1.682
1.677
0.003
0.006
16.1
15.0
0
0.31
0.34
0.31
1.641
1.627
1.586
0.003
0.006
0
0.31
0.32
0.32
0.009
1.582
1.568
1.555
0.041
0.003
0.006
SEM
Significance
Protein
Calsporin
NSC
NS
NS
NS
NS
NS
Protein × Calsporin
AEgg samples were taken at the tenth week of the experiment.
BBody weight was determined at the termination of study.
CNot significant at P > 0.05.
measure of Calsporin concentration in the
diets, monitor the effect of Calsporin level on
intestinal microflora, and determine the respec-
tive release of microbes in feces from each
Calsporin level [28]. This study was conducted
in an environmentally controlled facility where
daily temperatures were maintained at approxi-
mately 25.5°C except for a few days when
unusually high summer temperatures occurred.
To determine hen performance, mortality was
recorded daily, feed consumption (FC) and EP
were measured weekly, and egg weight (EW)
and egg specific gravity (SG) [29] were deter-
mined biweekly from a 2-d collection of eggs
during the week. Eggshell thickness [30] and
body weight measured at the termination of
study. All data were analyzed statistically [31]
to determine the effect of Calsporin and protein
level on hen performance.
RESULTS AND DISCUSSION
Egg SG
Egg SG is significantly related to percent-
age of cracks and is useful in estimating egg-
shell quality [29]. Calsporin level had an inter-
action (P < 0.01) with protein level on SG
during the 10-wk study (Table 2). The SG in-
creased more in hens fed the higher Calsporin
level (0.006%) than in hens fed the lower level
(0.003%) when dietary protein was 15%. This
effect of Calsporin level was reversed as di-

SOHAIL ET AL: LAYER RESPONSE TO CALSPORIN
383
TABLE 4. Influence of Calsporin on feed consumption of Hy-Line W36 hens (second cycle, 69 to 79 wk of age)
Feed consumption (g)
Week
Treatment
1
2
3
4
5
6
7
8
9
10
Mean
Protein (%)
17.3
16.1
70.2a 77.3a 87.1a 91.3a 91.9a 92.0a 91.2a 88.8a 93.7a 87.4a 87.0a
69.2a 74.9a 83.7b 88.2b 88.7b 89.9ab 88.9ab 86.6ab 90.9b 85.9ab 84.7b
68.4a 74.8a 82.4b 86.4b 87.2b 88.3b 87.6b 84.8b 90.0b 84.4b 83.4b
15.0
SEM
0.60
0.76
0.73
0.71
0.77
0.81
0.81
0.75
0.62
0.63
0.56
Calsporin (%)
0
69.5
74.8
84.7
88.5
88.9
89.4
88.5
86.0
91.1
85.1
84.6
0.003
0.006
SEM
68.3
70.0
0.60
75.1
77.1
0.76
84.0
84.5
0.73
88.3
89.0
0.71
89.1
89.8
0.77
90.1
90.7
0.81
89.3
90.0
0.81
86.5
87.2
0.75
91.1
92.4
0.62
85.8
86.8
0.63
84.8
85.7
0.56
Protein (%)
17.3
×
Calsporin (%)
0
71.5
69.2
69.9
77.1
77.3
77.5
88.0
86.9
86.4
91.0
91.9
90.9
90.8
92.3
92.5
90.5
92.2
93.3
90.0
92.2
91.3
87.1
89.2
88.8
93.2
93.8
94.2
86.1
88.5
87.5
86.5
87.4
87.2
0.003
0.006
16.1
15.0
0
67.8
68.4
71.4
72.8
74.7
77.3
83.9
82.8
84.4
88
87.2
89.3
88.7
88.5
89.0
89.8
89.6
90.2
88.7
89.1
88.9
86.8
86.5
86.5
91.2
90.6
90.8
86.2
85.9
85.6
84.4
84.3
85.3
0.003
0.006
0
69.1
67.4
68.6
74.5
73.4
76.4
82.2
82.3
82.8
86.5
85.8
86.9
87.1
86.4
88.1
87.9
88.4
88.7
86.8
86.5
89.6
84.1
83.9
86.3
88.9
89.0
92.1
83.0
82.9
87.4
83.0
82.6
84.7
0.003
0.006
SEM
1.04
1.31
1.26
1.23
1.33
1.40
1.40
1.29
1.07
1.08
0.98
Significance
Protein
Calsporin
NSA
NS
NS
NS
0.08
NS
***
NS
NS
***
NS
NS
***
NS
NS
**
NS
NS
**
NS
NS
**
NS
NS
***
NS
NS
**
NS
0.06
***
NS
NS
Protein × Calsporin
a,bMeans within a column and treatment section having different superscripts are significantly different (P < 0.05).
ANot significant at P > 0.05.
**P < 0.01.
***P < 0.001.
etary protein was increased to 16.1%, and SG
increased more in hens fed the lower level
(0.003%) than fed the higher level (0.006%).
The Calsporin level effect on SG was lost when
dietary protein was further increased to 17.3%.
This finding indicated that dietary protein level
influenced the effect of Calsporin on SG. In-
creasing dietary protein from 16.1 to 17.3%
had an adverse effect on SG of hens (P < 0.01),
which was expected due to increase in EW.
On average, SG decreased from 1.0816 to
1.0808 as protein level increased from 16.1
to 17.3%.
Eggshell Thickness
Dietary Calsporin or protein had no sig-
nificant effect (P > 0.05) on eggshell thickness
(Table 3). However, trends similar to that of
SG were observed when eggshell thickness
was measured.
Body Weight
Calsporin level or dietary protein had no
significant effect (P > 0.05) on body weight of
hens, and there were no Calsporin × protein
interactions (Table 3).

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JAPR: Research Report
TABLE 5. Influence of Calsporin on egg production of Hy-Line W36 hens (second cycle, 69 to 79 wk of age)
Egg production (%)
week
Treatment
1
2
3
4
5
6
7
8
9
10
Mean
Protein (%)
17.3
16.1
10.8a 39.0a 54.9a 70.5a 80.4a 82.9a 82.3a 82.1a 81.7a 82.7a 66.7a
7.8b 33.9b 49.6b 67.5a 76.6b 80.1b 82.5a 82.0a 80.7a 81.2a 64.2b
7.1b 31.2b 46.7b 62.4b 72.1c 76.6c 79.1b 79.2a 78.3b 78.7b 61.2c
15.0
SEM
0.94
1.54
1.52
1.25
1.06
0.85
0.78
0.94
0.82
0.82
0.65
Calsporin (%)
0
7.7
9.2
8.6
33.7
33.6
36.3
48.9
49.8
52.1
67.9
66.4
66.0
76.9
76.0
75.9
79.4
80.6
79.3
81.6
81.8
80.5
81.4
81.5
80.4
80.1
91.4
79.2
82.0
81.8
78.8
64.0
64.2
63.7
0.003
0.006
SEM
0.94
1.53
1.51
1.25
1.05
0.85
0.78
0.94
0.81
0.81
0.65
Protein (%)
17.3
×
Calsporin (%)
0
8.9
12.4
11.3
39.0
37.8
40.5
55.1a 71.0
55.0a 71.2
54.5a 69.0
80.9
82.3
77.4
82.8
85.2
80.1
82.1
84.1
80.3
83.5
83.6
78.3
82.8
83.3
78.5
84.2
83.7
79.5
67.0
67.9
64.9
0.003
0.006
16.1
15.0
0
6.3
8.2
8.7
30.7
33.2
37.0
44.8b 68.4
49.7ab 66.0
53.5a 68.1
77.4
74.2
78.0
79.5
78.2
82.0
83.2
81.2
83.0
82.5
80.4
82.9
80.5
80.8
80.9
83.3
81.0
79.7
63.7
63.3
65.4
0.003
0.006
0
8.0
7.1
6.4
31.4
29.9
32.1
46.9a 64.2
44.6a 62.1
48.7a 61.0
72.6
71.6
72.1
75.9
78.5
75.5
79.5
80.1
77.7
78.1
80.4
79.1
77.0
80.1
77.8
78.6
80.5
77.0
61.2
61.5
60.7
0.003
0.006
SEM
0.63
2.64
2.61
2.17
1.82
1.46
1.34
1.63
1.41
1.40
1.11
Significance
Protein
Calsporin
**
***
NS
NS
***
NS
NS
***
NS
NS
***
NS
NS
***
NS
*
**
NS
NS
*
NS
0.07
**
NS
NS
**
**
NS
***
NS
NS
NSA
NS
Protein × Calsporin
a,b,cMeans within a column and treatment section having different superscripts are significantly different (P < 0.05).
ANot significant at P > 0.05.
*P < 0.05.
**P < 0.01.
***P < 0.001.
FC
EP
Consistent with previous research [35, 36,
Within 2 wk, dietary protein had a linear
37] EP linearly increased (P < 0.01) within 1
wk as dietary protein was increased (Table 5).
Increasing protein from 15.0 to 17.3% linearly
increased (P < 0.001) average EP from 61.2
to 66.7%, a net increase of 5.5%. Dietary Cal-
sporin, however, had no effect on EP (P >
0.05), and there were no interactions.
effect (P < 0.05) on FC of hens (Table 4). As
protein level increased from 15.0 to 17.3%,
average FC linearly increased (P < 0.001) from
83 to 87 g/hen per d. Reports on the effect of
dietary protein on FC of hens are inconsistent
[34, 35, 36, 37]. Some report an increase in
FC due to increase in the amount of dietary
protein [37], whereas others report no effect
[36, 38]. Calsporin had no effect (P > 0.05)
on FC of hens. However, a trend indicating
increased FC with increasing dietary Calsporin
level was observed within 2 wk.
EW
Increasing dietary protein had a linear ef-
fect (P < 0.01) on EW within 2 wk (Table 6).
This finding was in agreement with previous

SOHAIL ET AL: LAYER RESPONSE TO CALSPORIN
385
TABLE 6. Influence of Calsporin on egg weight of Hy-Line W36 hens (second cycle, 69 to 79 wk of age)
Egg weight (g)
Week
Treatment
2
4
6
8
10
Mean
Protein (%)
17.3
16.1
15.0
SEM
62.6a
61.4b
60.6b
0.38
63.7a
62.4b
61.9b
0.24
63.7a
62.4b
62.2b
0.25
63.0a
62.4ab
61.9b
0.23
63.0a
61.9b
62.0b
0.26
63.2a
62.1b
61.7b
0.21
Calsporin (%)
0
61.9
61.3
61.5
0.38
62.9
62.2
62.8
0.24
63.1
62.3
62.8
0.25
62.7
62.2
62.4
0.23
62.3
62.2
62.4
0.26
62.6
62.1
62.4
0.21
0.003
0.006
SEM
Protein (%)
17.3
×
Calsporin (%)
0
62.8
62.6
62.4
63.9
63.6
63.6
63.8
63.2
64.0
63.0
62.9
63.0
62.7
63.2
62.9
63.3
63.1
63.2
0.003
0.006
16.1
15.0
0
61.7
60.6
61.8
62.5
61.8
62.8
62.9
62.0
62.1
62.6
62.1
62.3
62.0
61.9
61.8
62.4
61.7
62.2
0.003
0.006
0
61.1
60.6
60.2
0.66
62.3
61.3
62.2
0.41
62.4
61.8
62.4
0.43
62.4
61.6
61.8
0.39
62.0
61.6
62.4
0.45
62.0
61.4
61.8
0.36
0.003
0.006
SEM
Significance
Protein
Calsporin
**
NS
NS
***
0.07
NS
***
NS
NS
**
NS
NS
**
NS
NS
***
NS
NS
Protein × Calsporin
a,bMeans within a column and treatment section having different superscripts are significantly different (P < 0.05).
ANot significant at P > 0.05.
**P < 0.01.
***P < 0.001.
research that increasing dietary protein in-
creases EW [36, 37, 39]. Increasing protein
level from 15 to 17.3% linearly increased aver-
age (P < 0.001) EW from 61.7 to 63.2 g. Cal-
sporin supplementation in the diet had no in-
fluence (P > 0.05) on EW, and there were no
Calsporin × protein interactions.
beginning and toward the end of the experi-
ment. The amount of Calsporin fed was directly
related to the amount of intestinal microflora
TABLE 7. Influence of dietary Calsporin on fecal
bacterial count
Fecal bacterial count
Mean (log cfu/g feces)
Fecal Bacterial Count and Hen Mortality
Fecal bacteria were counted to determine
the level of bacteria excreted relative to the
Calsporin level fed and to determine the accu-
racy of feeding Calsporin levels within each
treatment. Increasing each dietary Calsporin
increased (P < 0.001) the fecal bacterial count
(Table 7). This increase was observed at the
Calsporin (%)
Begin
End
Average
0.000
0.003
0.006
SEM
2.66c
5.12b
5.45a
0.07
2.71c
4.91b
5.25a
0.08
2.65c
4.98b
5.35a
0.05
a–cMeans within a column having different superscripts are
significantly different (P < 0.05).

386
JAPR: Research Report
excreted. This result suggests that Calsporin
had no adverse effect on the performance of
hens. These results also indicate that Calsporin
levels were correctly fed during the course of
this study, and measures taken to avoid con-
tamination among the treatments were success-
ful. There were no Calsporin × protein interac-
tions (not shown) nor was an effect of Cal-
sporin or dietary protein on mortality of hens
observed.
CONCLUSIONS AND APPLICATIONS
1. Calsporin (Bacillus subtilis C-3102) level had a significant interaction with dietary protein
level on egg SG.
2. Further research is needed to optimize any potential benefit of using Calsporin relative to
dietary protein in commercial hen diets to improve hen performance and eggshell quality.
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387
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