Research Note
Guar Meal Diets as an Alternative Approach to Inducing Molt and Improving
Salmonella Enteritidis Resistance in Late-Phase Laying Hens
O. Gutierrez,1 C. Zhang, D. J. Caldwell, J. B. Carey, A. L. Cartwright, and C. A. Bailey
Department of Poultry Science, Texas A&M University, College Station, TX 77843-2472
ABSTRACT Induced molting of laying hens is a prac-
tice used by commercial egg producers to increase the
productive lifetime of their flock. However, the conven-
tional method of inducing molt, which involves removal
of feed, water, or both as well as a reduction in photope-
riod to less than a natural day has drawn criticism due
to animal welfare and food safety concerns. The objective
of this study was to explore the efficacy of diets containing
high levels of guar meal (GM) in inducing molt and reduc-
ing susceptibility to Salmonella Enteritidis colonization in
late-phase laying hens. Late-phase (68 wk old) Lohmann
laying hens were either full-fed standard laying hen diets
(nonmolted control), induced to molt by feed withdrawal,
or full-fed standard laying hen diets containing 20% GM
with or without 250 units/kg of mannanase Hemicell
Key words: molt, laying hen, guar, Salmonella
INTRODUCTION
Induced molting of late-phase laying hens is used by
many commercial egg producers in the United States to
stimulate multiple laying cycles and restore egg quality.
Conventional methods of inducing molt involve fasting
and in some cases removing water for a sufficient period
of time to completely regress the tissues of the reproduc-
tive tract. However, using feed withdrawal to induce
molt has drawn criticism due to concerns about the hu-
mane treatment of animals used for food production.
Additionally, stress associated with long-term feed
withdrawal impairs the immunological function of lay-
ing hens and increases the susceptibility of layers to
Salmonella infection.
Several alternative methods to molting hens by feed
withdrawal have evaluated the efficacy of full-feeding
diets with a variety of mineral imbalances, including
low-sodium diets (Berry and Brake, 1985), diets deficient
in calcium (Martin et al., 1973), and diets high in zinc
©2008 Poultry Science Association Inc.
Received August 13, 2007.
Accepted November 24, 2007.
1
Corresponding author: ogutierrez@tamu.edu
536
supplementation. On the fourth day of treatment, all hens
were orally challenged with SE (1.65 × 107 cfu). Hens
were killed and evaluated for Salmonella colonization and
differences in organ weights 5 d postinoculation. Salmo-
nella Enteritidis present in crop, liver, ovary, and cecal
contents were significantly reduced by feeding GM with
enzyme supplementation compared with feed with-
drawal hens. No significant differences were observed in
reproductive tract weights of molted groups, although a
difference in liver weight was detected. Results indicate
that feeding diets containing 20% GM are as effective as
complete feed withdrawal with respect to inducing molt
with the added benefit of improved resistance to Salmo-
nella Enteritidis colonization and translocation.
2008 Poultry Science 87:536–540
doi:10.3382/ps.2007-00337
(Berry and Brake, 1985). Another approach to inducing
molt in laying hens involves full-feeding grain and le-
gume by-products rich in indigestible plant fibers such
as alfalfa (Donalson et al., 2005), cottonseed meal (Davis
et al., 2002), and guar meal (GM; Patel and McGinnis,
1981; Zimmermann et al., 1987). In addition to inducing
molt, the use of wheat middlings (Seo et al., 2001) is
also effective in reducing the incidence of Salmonella
Enteritidis in eggs and internal organs.
Mannan oligosaccharides are believed to bind fim-
briae present on the extracellular membrane of Salmo-
nella cells, limiting their ability to bind and colonize
intestinal epithelial cells (Fernandez et al., 2002). Guar
(Cyamopsis tetragonoloba) meal, a by-product of guar gum
processing, contains 18 to 20% gum residue (Bakshi et
al., 1965; Nagpal et al., 1971). Guar gum is a linear poly-
saccharide of β-galactomannan consisting of a 1→4-
linked β-D-mannopyranose backbone with branched
1→6-α-D-galactopyranose. Partially hydrolyzed guar
gum (PHGG), produced by the hydrolysis of guar gum
by β-endomannanase, is comprised of neutral polysac-
charides consisting of a mannose backbone chain with
single galactose side units occurring on approximately
2 out of every 3 mannose units.
Guar gum has been reported to elicit protective effects
against the colonization of pathogenic bacteria within
 RESEARCH NOTE 537
the intestinal tract of rats (Noack et al., 1998). Ishihara et Table 1. Composition of nonmolted control diet and molt-inducing
diets containing 20% guar meal (GM)
al. (2000) found that pullets and laying hens consuming
PHGG had a decreased incidence of colonization by Nonmolted 20% GM
Salmonella Enteritidis in the organs and intestinal tract, Ingredients (%) control1 diets2
with a concurrent increase in excretion of Salmonella Corn 75.84 72.29
Enteritidis into the feces. The same group also reported GM3 0.00 20.00
Dehulled soybean meal 16.70 0.00
a decreased incidence of SE in egg components when DL-Met 0.16 0.10
PHGG was administered in the diet. These results sug- L-Lys HCl 0.04 0.21
gest that the administration of guar gum or PHGG may Fat (animal-vegetable blend) 0.52 0.59
Limestone 4.51 4.52
prevent Salmonella Enteritidis colonization in pullets and Monodicalcium phosphate 1.46 1.48
laying hens. Salt 0.47 0.51
The purpose of this experiment was to evaluate the Trace minerals4 0.05 0.05
Vitamins5 0.25 0.25
use of GM, with and without enzyme treatment (Hemi-
1
cell, ChemGen Corp., Gaithersburg, MD), as an alterna- Calculated analysis of all diets containing 20% GM was as follows:
CP, 14.06%; ME, 2,893 kcal/kg; Ca, 2.00%; available P, 0.40%; Met,
tive method of inducing molt and improving resistance 0.32%; Lys, 0.68%; Thr, 0.42%; and Trp, 0.13%.
to Salmonella infection in late-phase laying hens. Tissue 2
Calculated analysis of nonmolted control diet was as follows: CP,
regression rates of certain organs were measured in 14.68%; ME, 3,000 kcal/kg; Ca, 2.01%, available P, 0.40%; Met, 0.40%;
molted laying hens. Lys, 0.72%; Thr, 0.54%; and Trp, 0.16%.
3
The nutrient matrix used for GM was as follows: CP, 38.3%; ME,
2,033 kcal/kg; Ca, 0.16%; available P, 0.16%; Met, 0.45%; Lys, 1.64%;
MATERIALS AND METHODS Arg, 4.90%; Thr, 1.04%; and Trp, 0.43%.
4
Trace minerals premix added at this rate yields the following: 27.5
Experimental Design mg of S, 150 mg of Mn, 16.8 mg of Fe, 1.7 mg of Cu, 125.5 mg of Zn,
0.25 mg of Se, 1.05 mg of I, 0.84 mg of Mb molybdenum per kilogram
and Molting Procedure of diet.
5
Forty-three Lohmann White late-phase laying hens Vitamin premix added at this rate yields the following: 11,023 IU of
vitamin A, 46 IU of vitamin E, 3,850 IU of vitamin D3, 1.47 mg of vitamin
(68 wk old) of similar BW (1,527 ± 114 g) were wing- K, 2.94 mg of thiamin, 5.85 mg of riboflavin, 20.21 mg of pantothenic
banded and randomly allocated into 9 cages of a rearing acid, 0.55 mg of biotin, 1.75 mg of folic acid, 477.67 mg of choline, 16.5
battery housed in an environmentally controlled room. ␮g of vitamin B12, 45.93 mg of niacin, and 7.17 mg of pyridoxine per
kilogram of diet.
Eight cages had 5 hens each, and 1 cage had 3 hens,
which served as a negative control group for the Salmo-
nella Enteritidis colonization portion of this study (AUP
Laboratory (phage type 13A), was selected for resistance
2003-0256, approved by the Institutional Agricultural
to novobiocin (NO; No. n-1628, Sigma Chemical Co., St.
Animal Care and Use Committee, Texas A&M Univer-
Louis, MO) and to nalidixic acid (NA; No. n-4382, Sigma
sity, College Station, TX). Hens were allowed to accli-
Chemical Co.) within our laboratory. Salmonella Enteriti-
mate for 1 wk with free access to a typical corn-soy
dis was grown according to the method of Lee and Fal-
laying hen feed and water while on a 16L:8D lighting
kow (1990), allowing for attainment of log-phase
program. At the end of the acclimation period, hens
growth. Bacteria were washed 3 times in distilled water
were weighed individually. Four treatments, consisting
by centrifugation (3,000 × g) and spectrophotometrically
of a feed withdrawal group (FW), full-fed groups com-
quantified to a stock concentration of approximately 1
bining standard laying hen feed with either 20% GM
× 109 cfu/mL in distilled water using a standard curve
(20% GM) or 20% GM supplemented with 250,000 units/
generated from comparison of multiple spread platings
kg of β-mannanase (Hemicell; 20% + E), and a full-fed
standard laying diet group (nonmolted control) were and optical densities. Bacteria were then diluted to a
randomly assigned to 8 cages. Nutrient composition of challenge concentration of 1.65 × 107 cfu/mL as deter-
GM (Table 1) was previously determined by Conner mined by multiple spread platings.
(2002) with amino acid analysis by Degussa-Huls Corpo-
ration (Applied Technology Chemical Group, Allen- Organ Weights and Salmonella Recovery
dale, NJ).
Five days postinoculation (d 9 of treatment), all hens
Beginning on the first day of treatment, the light pro-
were euthanized by CO2 asphyxiation, and the crop,
gram was changed to 8L:16D. On d 4 of treatment, all
liver, spleen, ovary, oviduct, and ceca of each bird were
hens, with the exception of the 3 extra hens that served
excised aseptically as described below. After clamping
as a negative control group, were inoculated with 1 mL
of Salmonella Enteritidis (1.65 × 107 cfu/mL) by oral ga- across the pre- and postcrop esophagi using surgical
vage. Eggs laid during the study were collected and Carmalt forceps, the crop was sectioned aseptically with
recorded daily. All hens received free access to water. the lumen and contents intact and collected into individ-
ual Whirl-Pac bags. The whole liver, spleen, ovary, and
Salmonella Enteritidis Inocula oviduct (from infundibulum to shell gland) were excised
aseptically and collected into individual Whirl-Pac bags
A primary poultry isolate of Salmonella Enteritidis, separately and weighed. Twenty milliliters of tetrathio-
obtained from the USDA National Veterinary Services nate broth base (No. 0104-17-6, Difco Laboratories, De-
538 GUTIERREZ ET AL.
Table 2. Body weight reduction (BWR), days to 0 egg production, and absolute and relative organ weight of
laying hens molted by various methods1

Liver Spleen Ovary Oviduct

BWR2 Days to
Treatment (%) 0 egg3 Wt (g) % Wt (g) % Wt (g) % Wt (g) %
a d c b a b b b
FW control 27.01 5 24.2 2.08 1.55 0.13 10.6 0.90 17.7 1.53b
20% GM 19.14b 6 31.2c 2.46b 1.97a 0.16a 10.4b 0.83b 19.5b 1.55b
20% GM + E 16.10c 5 36.2b 2.76a 1.96a 0.15a 10.5b 0.80b 20.6b 1.57b
Nonmolted 0.07d — 48.1a 2.99a 1.56b 0.10b 45.7a 2.85a 65.2a 4.07a
Pooled mean square error 3.50 — 4.9 0.30 0.45 0.03 4.4 0.28 5.7 0.42
a–d
Means in the same column lacking a common superscript were significantly different (P ≤ 0.05).
1
Experimental laying hens were 69 wk old at the beginning of the experiment.
2
Inital BW were 1,613, 1,571, 1,561, and 1,611g for feed withdrawal (FW), 20% guar meal (GM), 20% GM +
250,000 units/kg of β-mannanase (E), and nonmolted groups, respectively. Final BW were determined on d 9
of the trial.
3
Days needed to cease laying for each treatment were not subjected to statistical analysis.

troit, MI) was added into each Whirl-Pac bag. Crop and
oviduct samples were stomached (Tekmar Stomacher
80, Laboratory Blender, Cincinnati, OH) for 60 s. One
cecum from each hen was aseptically excised, minced,
and collected into 50-mL conic centrifuge tubes with 30
mL of tetrathionate broth base and vortexed for 30 s.
The other cecum was aseptically sectioned, and approxi-
mately 0.5 g of cecal content was squeezed into a centri-
fuge tube containing 4.5 mL of Butterfield’s buffer solu-
tion then vortexed until the cecal contents were dis-
persed homogeneously. The solution was serially
diluted to a final concentration of 10−4 by sequentially
transferring 0.5 mL to another centrifuge tube con-
taining 4.5 mL of buffer solution. Each dilution (0.1 mL)
was plated onto a brilliant green agar (BGA; No. 0285-
01-5, Sigma Chemical Co.) plate containing NO 25 ␮g/
mL and NA 20 ␮g/mL (NO-NA-BGA) to prohibit
growth of Salmonella other than the antibiotic-resistant
challenge isolate. Plates were incubated for 24 h at 37°C,
and Salmonella Enteritidis number (cfu/g of cecal con-
tents) was determined. Cecal contents in which Salmo-
nella Enteritidis was not detected at the 10−1 dilution on
BGA plates were scored as 0 cfu. The remaining organ
samples were stored at 0°C for 108 h before incubation
for 24 h at 37°C. After the enrichment phase, each sample
was individually streaked onto a NO-NA-BGA plate,
incubated for another 24 h at 37°C, and examined for
the presence of Salmonella Enteritidis.
Statistical Procedure
Body weight reduction and absolute and relative
weights of organs were subjected to ANOVA by the
GLM procedure of the SAS System (SAS System for
Windows, Release 8.1, SAS Institute Inc., Cary, NC) for
a completely randomized design. Each hen served as an
individual experimental unit. Feed consumption data
were not collected. The presence of Salmonella Enteritidis
in each organ was analyzed by Fisher’s exact test (Uiten-
broek, 2000). The P-value for the same or a stronger
association was used in Fisher’s test (Garson, 2004). The
total Salmonella Enteritidis-positive numbers of organs
from each group and from all hens were compared by
Pearson’s χ2 test. The number of Salmonella Enteritidis
colony-forming untis per gram of cecal content was first
subjected to log transformation and then analyzed using
the GLM procedure of the SAS System for a completely
randomized design. Data from the 3 extra hens were not
included when analyzing Salmonella Enteritidis presence
and log colony-forming unit data. Significance was ac-
cepted at P ≤ 0.05.
RESULTS
BW and Laying Activity
Mean BW of each group was similar at the beginning
of the trial but significantly diverged by the end of the
study (Table 2). All induced-molt treatment groups com-
pletely ceased egg production by the sixth day of treat-
ment, whereas nonmolted hens continued laying
throughout the 9-d study.
Absolute and Relative Organ Weight
Liver weights of all groups were significantly different
from each other. However, relative liver weight to BW
for nonmolted hens and hens fed 20% GM + E were not
different, and both were proportionally larger than the
relative liver size of FW and 20% hens (Table 2). Non-
molted hens had significantly lower relative spleen
weights than molt-induced hens, which were not differ-
ent from each other. Nonmolted hens, which continued
laying throughout the study, had significantly higher
absolute and relative weights of ovary and oviduct than
molted hens. Differences were not observed among the
molt-induced groups.
Salmonella Enteritidis Colonization
and Organ Invasion
Salmonella Enteritidis was not detected in the organ
samples or cecal contents of nonchallenged negative
control hens (Table 3). Compared with nonmolted con-
trol hens, all molt-induced hens had a higher incidence
of Salmonella Enteritidis in cecum and oviduct with no
 RESEARCH NOTE 539
Table 3. Colonization and invasion of Salmonella Enteritidis into organs and Salmonella Enteritidis colony-forming
units in cecal contents of laying hens induced to molt by various methods1

Salmonella Enteritidis-positive hens/total hens Log Salmonella

Enteritidis cfu/g
2
Treatment Hens (n) Crop Liver Spleen Ovary Oviduct Ceca All organs of cecal content
a a ab a a a
FW control 10 7/10 9/10 3/10 7/10 8/10 10/10 44/60a 6.717a
20% GM 10 0/10b 6/10a 5/10a 3/10ab 5/10a 9/10a 28/60b 3.457b
20% GM + E 10 0/10b 1/10b 1/10ab 1/10b 5/10a 9/10a 17/60c 2.955b
Nonmolted 10 2/10b 0/10b 0/10b 0/10b 0/10b 2/10b 4/60d 1.270c
Nonchallenged 3 0/3 0/3 0/3 0/3 0/3 0/3 0/18 ND3

Means in the same column lacking a common superscript were significantly different (P ≤ 0.05).
a–d
1
Experimental laying hens were 70 wk old at the time of sampling.
2
FW = feed withdrawal; GM = guar meal; E = 250,000 units/kg of β-mannanase.
3
Data from nonchallenged hens were not included in the Salmonella Enteritidis colony-forming unit analysis.

difference between molting methods. In liver samples,
FW hens had higher incidences of SE than 20% GM +
E and nonmolted control hens. The FW hens also had
increased susceptibility to Salmonella Enteritidis in the
crop and ovary than nonmolted hens and hens fed GM +
E. More pronounceable differences between treatments
were detected when the incidence of Salmonella Enteriti-
dis colonization was summed across all organ samples.
All molted hens had higher total Salmonella Enteritidis-
positive numbers in the 6 tested organs than nonmolted
hens. Among molt-induced groups, FW hens had a
higher prevalence of Salmonella Enteritidis than hens fed
20% GM, which in turn were higher than hens fed 20%
GM + E. With respect to colony-forming units of Salmo-
nella Enteritidis in cecal contents, FW hens contained
cecal populations more than 3 logs higher than those of
either group of hens fed GM, which were 1.5 to 2 logs
higher than that of nonmolted hens.
DISCUSSION
A reduction in BW of molted hens ranging from 25
to 35% is recommended to achieve optimal involution
of the reproductive tract and subsequent postmolt egg
production levels (Zimmermann et al., 1987; Carey and
Brake, 1989). In this experiment, however, hens fed 20%
GM + E experienced a BW loss of only 16%, whereas hens
consuming 20% GM without enzyme supplementation
showed a reduction in BW of 19%. Molted hens had
losses in total weight of oviduct, ovary, and liver ranging
from 92 to 107 g and losses due to regression of other
tissues ranging from approximately 110 to 340 g. Hens
molted by the 20% GM diets lost equal amounts of
weight from the reproductive tract and less weight in
other tissues and organs than FW hens. Therefore, the
BW loss occurring exclusive of the reproductive tract in
FW hens may not be critical to molt induction. This
finding suggests that the recommendation of a reduction
in BW by 25 to 35% may not be suitable when a full-
feeding alternative molting technique is used.
In the present study, hens molted by 20% GM diets
had significantly reduced Salmonella Enteritidis coloni-
zation numbers in most organs, and the GM diet with
enzyme supplementation had a stronger effect on Salmo-
nella Enteritidis resistance than the GM diet without
enzyme relative to fasted control groups. Evidence of
the ability of GM to enhance Salmonella Enteritidis resis-
tance of molted hens also was supported by the more
than 3 log reduction of Salmonella Enteritidis colony-
forming units of cecal contents relative to the effects
of FW. Consistency between the occurrence of positive
Salmonella Enteritidis organ samples and Salmonella En-
teritidis colony-forming units per gram of cecal contents
indicates that induced molting by full-fed GM diets im-
proves resistance to Salmonella Enteritidis in molted
hens.
With respect to days to 0 egg production, this study
demonstrates that full-feeding late-phase laying hens
with 20% GM with and without β-mannanase (Hemicell)
appears to be as effective in inducing molt as the conven-
tional FW method and is potentially more desirable with
respect to animal welfare concerns due to a presumed
decrease in stress associated with BW loss. Additionally,
laying hens induced to molt by GM feeding exhibit im-
proved resistance to Salmonella Enteritidis colonization
when compared with hens molted by complete feed
withdrawal. Furthermore, supplementation of β-man-
nanase (Hemicell) to diets containing high levels of GM
appears to enhance resistance to Salmonella Enteritidis
colonization in molted laying hens.
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