J. J. Villalba, A. Bach and I. R.

Ipharraguerre
Feeding behavior and performance of lambs are influenced by flavor diversity
doi: 10.2527/jas.2010-3435 originally published online March 31, 2011
2011, 89:2571-2581. J ANIM SCI
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Feeding behavior and performance of lambs are influenced
by flavor diversity
1,2
J. J. Villalba,*
3
A. Bach, and I. R. Ipharraguerre
*Department of Wildland Resources, Utah State University, Logan 84322-5230; Instituci Catalana
de Recerca i Estudis Avanats (ICREA) and Institut de Recerca i Tecnologia Agroalimentries (IRTA),
Ruminant Production, Caldes de Montbui 08140, Spain; and Lucta, S.A. Montorns del Valls 08170, Spain
ABSTRACT: This study determined whether early
experiences of sheep with the same feed, but presented
in multiple or single flavors, would influence intake, the
profile of hormones involved in feed intake regulation,
and the subsequent acceptability of novel feeds. Thirty-
five 2-mo-old lambs were randomly assigned to 5 treat-
ments (7 lambs/treatment). Lambs in 1 treatment (the
diversity treatment) were simultaneously fed an unfla-
vored plain ration of alfalfa (control) and barley (75:25;
as-fed basis) and the same ration mixed (0.2%) with 1
of 3 flavors: 1) sweet, 2) umami, or 3) bitter. The other
4 treatments (monotonous diets) received only 1 of the
4 rations. All animals were fed their respective rations
from 0800 to 1600 h for 60 d. On d 55, intake was re-
corded every 30 min for 8 h. On d 58, blood samples
from lambs were collected at 1 h prefeeding and at
30, 60, 210, 300, and 540 min postfeeding. Preference
tests were conducted by simultaneously offering novel
feeds: 1) high-energy feed, 2) high-protein feed, 3) beet
pulp mixed with phytochemicals, or 4) low-quality feed.
Lambs in the diversity treatment consumed more feed
than did lambs in the other treatments (P < 0.001).
Lambs in the diversity treatment consumed equiva-
lent amounts of plain and umami feeds, with a greater
amount (P < 0.001) of the umami feed being consumed
than the bitter and sweet feeds. Lambs in the diversity
treatment tended to grow faster than did lambs in the
other treatments (P = 0.06). On d 55, lambs in the di-
versity treatment showed decreased (P < 0.05) feed in-
take compared with lambs in the other treatments dur-
ing the 2 peaks of food consumption (30 and 270 min
from feeding) and showed a trend for the least plasma
concentrations of ghrelin (P = 0.06). In contrast, lambs
in the diversity treatment consumed more feed than
did lambs exposed to monotonous flavors at 60, 90,
120, and 180 min from feeding (P < 0.05). Lambs in
the diversity treatment also showed the least concentra-
tions of cholecystokinin and glucagon-like peptide 1 (P
< 0.001). There were trends for the greatest concen-
trations of leptin (P = 0.14) and IGF-1 (P = 0.16) in
the diversity treatment, and for the least concentration
of leptin in the bitter treatment (P = 0.14). Previous
experience with flavored feeds affected the preference
of lambs for high-energy and low-quality feeds, and for
beet pulp mixed with phytochemicals (treatment
feed day effect; P < 0.05). Thus, exposure to diverse
flavors has the potential to increase feed intake and
induce a more even consumption of feed across time
by reducing peaks and nadirs of intake compared with
exposure to monotonous rations. Flavor diversity may
also influence the initial acceptability of and preference
for novel feeds.
Key words: diversity, eating pattern flavor, hormone, intake regulation, sheep
2011 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2011. 89:25712581
doi:10.2527/jas.2010-3435
INTRODUCTION
Ruminants evolved in diverse environments, consum-
ing arrays of feeds of different chemical and physical
characteristics (Provenza et al., 2007). Nevertheless,
current intensive feeding systems are characterized
by feeding animals monotonous diets. Evidence from
rats and humans indicates that a repeated presenta-
tion of the same feed may result in a reduction in feed
intake (McSweeney and Swindell, 1999). At meal ini-
tiation, chemical senses (i.e., smell and taste) generate
oro-sensorial signals that trigger satiety (Blundell et
1
This research was supported by grants from Lucta S.A. (Mon-
torns del Valls, Spain) and the Utah Agricultural Experiment
Station (Logan). This paper is published with the approval of the
Director, Utah Agricultural Experiment Station, and Utah State
University, as journal paper number 8234.
2
We acknowledge R. Stott (Utah State University, Logan) for vet-
erinary services.
3
Corresponding author: juan.villalba@usu.edu
Received August 16, 2010.
Accepted March 17, 2011.
2571
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al., 1994) through a decline in wanting (related to ap-
petite) or liking (related to pleasure, palatability, or
hedonism) of the consumed feed. This process, known
as sensory-specific satiety (Rolls et al., 1982), has been
shown to play a role in regulating feed consumption
in humans (Srensen et al., 2003). In contrast, diverse
oro-sensorial stimuli may restore the motivation to eat
(Epstein et al., 2009) by enhancing feed acceptability
(Rolls, 1979; Treit et al., 1983). This effect may lead
to changes in feeding frequency and in the hormonal
profiles involved in feed intake regulation (Bello et al.,
2009). Furthermore, offering feeds in various flavors to
postpubertal heifers altered short-term feed preferences
(Atwood et al., 2001). Because experiences during de-
velopment induce lifelong neurological, morphological,
or physiological changes (Provenza and Villalba, 2006),
providing oro-sensorial diversity early in life may have
more pronounced effects than doing so at later stages
of life (Nolte and Provenza, 1992).
The hypothesis of this study was that providing oro-
sensorial diversity to ruminants after weaning would
alter feeding behavior, resulting in enhanced feed in-
take and animal performance. To test this hypothesis,
lambs were fed the same diet in single (monotonous) or
multiple (diversity) flavors, and changes in feed intake,
BW, feeding pattern, some appetite-controlling hor-
mones, and preferences for novel feeds were monitored.
MATERIALS AND METHODS
The study was conducted at the Green Canyon Ecol-
ogy Center, located at Utah State University in Logan,
according to procedures approved by the Utah State
University Institutional Animal Care and Use Commit-
tee.
Animals and Dietary Treatments
During the study, 35 commercial Finn-Columbia-
Polypay-Suffolk crossbred lambs of both sexes (2 mo
of age) with an average initial BW of 25 1 kg were
individually penned outdoors, under a protective roof,
in individual, adjacent pens measuring 2.4 3.6 m.
Throughout the study, lambs had free access to fresh
water and trace mineral salt blocks. Lambs were famil-
iar with alfalfa and barley grain because these feeds
constituted the basal diet of their mothers. Animals
were weaned and introduced into their individual pens
and were fed alfalfa pellets ad libitum and 300 g/d of
barley grain for 12 d until the beginning of the study.
Lambs were randomly divided into 5 treatments (7
lambs/treatment) and conditioned with different senso-
rial experiences. Lambs in 1 treatment (diversity) were
simultaneously fed an unflavored ration (plain) of al-
falfa and barley [75:25; 2.9 Mcal/kg; 15% CP (AOAC,
2002)] and the same ration presented in 3 different non-
nutritive flavors (0.2%): 1) sweet, 2) umami, and 3) bit-
ter (Lucta, S.A., Montorns del Valls, Spain). Lambs
in the other 4 treatments (monotonous diets) received
only 1 of the 3 flavored feeds or the plain diet through-
out the study. All lambs had ad libitum access to their
respective treatment diets from 0800 to 1600 h for 60
d. Additional feed was added at 1200 h when refusals
were below 10% of the amount initially offered. Daily
refusals were weighed, and daily intake was expressed
per kilogram of metabolic BW. Lambs were weighed on
d 1, 33, and 60 of the study (Table 1).
Feeding Behavior and Blood Sampling
On d 55, lambs were fed at 0800 h and feed intake
was measured every 30 min for 8 h as an estimate of
feeding behavior throughout the day (Table 1). On d
56, all lambs were fed at 0800 h and 1 observer recorded
eating behavior using the scan sampling method de-
scribed by Altman (1974; Table 1). Scan samples were
taken at 5-min intervals for a period of 8 h to record
bouts of feeding and inactivity. Frequency of feeding
was calculated as a percentage of the total number of
scans recorded. On d 57, three randomly chosen ani-
mals within each treatment were fitted with indwelling
catheters in the left jugular vein (1.4 mm i.d., 1.7 mm
o.d.; Abbott Laboratories, Abbott Park, IL). On d 58,
10 mL of blood was collected into heparinized tubes
Table 1. Chronology, feeds offered, and measurements in the study
Day Feeds offered Measurement
1 to 60 A plain ration of alfalfa and barley (75:25; plain treatment);
the same ration mixed with sweet, umami, or bitter flavors;
or a simultaneous offer of the 4 flavored feeds (diversity
treatment)
Feed intake from 0800 to 1600 h (d 1 to 60); feed intake
measured every 30 min from 0800 to 1600 h (d 55); scan
samples at 5-min intervals from 0800 to 1600 h (d 56); blood
extractions for hormone analyses (d 58); BW (d 1, 33, and 60)
61 to 105 Ad libitum intake of alfalfa pellets
106 to 109 A simultaneous offer of feeds high in energy
1
Feed intake from 0800 to 0815 h
110 to 113 A simultaneous offer of feeds high in protein
2
Feed intake from 0800 to 0815 h
114 to 117 A simultaneous offer of phytochemical-enriched feeds
3
Feed intake from 0800 to 0815 h
118 to 121 A simultaneous offer of feeds of low quality
4
Feed intake from 0800 to 0815 h
1
Oats, milo, beet pulp, and corn.
2
Wheat gluten meal, rabbit pellets, Calf-Manna (Manna Pro Products, Chesterfield, MO), and soybean meal.
3
Beet pulp mixed with condensed quebracho tannin powder (10%), quillaja bark saponin powder (2%), and sagebrush terpenes (1.8% camphor,
1.1% 1,8-cineole, and 0.1% p-cymene).
4
Wheat straw, wheat bran, and grape pomace.
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(Freeze-Dried Sodium Heparin, 143 USP units, BD Va-
cutainer, Franklin Lakes, NJ) from catheters at 1 h
prefeeding and at 30, 60, 210, 300, and 540 min post-
feeding. Samples were immediately centrifuged at 3,000
g for 15 min at 4C to harvest plasma, and samples
were subsequently stored at 30C until analyses for
ghrelin, insulin, leptin, IGF-1, glucagon-like peptide 1
(GLP-1), and cholecystokinin (CCK). Insulin deter-
minations were conducted using RIA with an RI-13K
kit (Millipore, Bedford, MA) with intra- and interassay
CV of 3.0 and 9.0%, respectively. Leptin was also ana-
lyzed by RIA using an XL-85K multispecies kit (Milli-
pore) with resulting intra- and interassay CV of 7.6 and
7.8%, respectively. Insulin growth factor-1 was deter-
mined using ELISA with an AC-27F1 kit (IDS, Foun-
tain Hills, AZ) with intra- and interassay CV of 2.0 and
3.4%, respectively. Ghrelin was analyzed by RIA with
an R90 kit (Mediagnost, Reutlingen, Germany) with
intra- and interassay CV of 9.3 and 6.8%, respectively.
Cholecystokinin was also determined using RIA with
an RB302 kit (IBL International, Hamburg, Germany)
with intra- and interassay CV of 2.5 and 8.9%, respec-
tively. Likewise, GLP-1 was determined by RIA with an
RK-028-13 kit (Phoenix Pharmaceuticals Inc., Burlin-
game, CA) with an intraassay CV of 4.0%.
Preference Tests for Novel Feeds
On d 61, all animals were offered alfalfa pellets for ad
libitum intake for 45 d (Table 1). During this washout
period all animals had the same basal diet and flavor
experiences before being tested for their preferences for
novel feeds. The aim was to ensure that differences in
feeding responses to novel feeds could be attributed to
the treatments that animals experienced early in life
and not to the short-term effects of flavors consumed
before testing.
Four series of tests were conducted to determine
whether different experiences with flavors (i.e., diverse
or monotonous diets) after weaning affected the pref-
erence and acceptance of novel feeds (Table 1). The
intake and preference for each feed were determined
by simultaneously offering all tested feeds ground to
1- to 2-mm particle size to all lambs for 15 min at
0800 h to establish the initial neophobic response of the
animals to the feeds. Subsequently, all lambs had ac-
cess to alfalfa pellets until 1600 h. Preference tests were
conducted for 4 consecutive days. In test 1, lambs were
presented with feeds high in energy (whole oats, whole
milo, beet pulp, and corn); in test 2, they received feeds
high in protein [wheat gluten meal, rabbit pellets, Calf-
Manna (Manna Pro Products, Chesterfield, MO), and
soybean meal]; in test 3, animals were offered beet pulp
mixed with phytochemicals [condensed quebracho tan-
nin powder (10%), quillaja bark saponin powder (2%),
and sagebrush terpenes (Sigma, St. Louis, MO; 1.8%
camphor, 1.1% 1,8-cineole, and 0.1% p-cymene mixed
in 4% vegetable oil)]; and in test 4, lambs had access
to feeds of low quality (wheat straw, wheat bran, and
grape pomace; Table 2). The study began on May 26,
2009 (dietary treatments), and ended on October 4,
2009 (preference tests for novel feeds).
Statistical Analyses
Feed intake (as-fed basis; g/kg of BW
0.75
), ADG,
eating events (as percentages of the total number of
scans), plasma hormone concentrations, and preference
for novel feeds [(intake of feed/total intake) 100] dur-
ing the preference tests were analyzed as a split-plot
design with lambs (random factor) nested within treat-
ment. Treatment (diversity, sweet, umami, bitter, or
plain) was the between-animal factor, novel feed (pref-
erence tests for novel feeds) was the within-animal fac-
tor, and time (eating behavior) or day (feed intake) was
the repeated measure in the analyses (fixed factors).
The fixed effect of sex was included in the model but
was later removed because of a lack of significant ef-
fects (P > 0.10). Separate analyses were conducted for
the diversity treatment to estimate lamb preferences
for each flavored feed. In this case, animal and flavored
feeds (sweet, umami, bitter, or plain) were the whole-
plot factors and day was the repeated measure. All
analyses were computed using a mixed-effects model
(SAS Inst. Inc., Cary, NC). The variance-covariance
structure used was the autoregressive order-1, which
yielded the smallest Bayesian information criterion.
Table 2. Metabolizable energy, digestible protein, and
crude fiber of novel feeds (DM basis) offered to 5 groups
of lambs exposed during a 60-d period to the same feed
offered in different flavors: plain, bitter, sweet, umami,
and diversity (a simultaneous offer of the 4 flavored
feeds)
Feed
Nutrient composition
ME,
Mcal/kg
DP,
%
Crude
fiber, %
Feeds high in energy
1

Whole oats 2.78 10.4 12.1
Whole milo 3.18 6.8 2.5
Beet pulp 2.78 6.7 16.5
Corn 3.15 6.5 2.2
Feeds high in protein
Wheat gluten meal
2
3.2 39.8 4.8
Rabbit pellets
3
2.2 14.6 15.2
Calf-Manna
4
2.5 25.0 6.0
Soybean meal
1
3.07 40.9 6.6
Feeds of low quality
Wheat straw
1
1.48 3.5 41.6
Wheat bran
1
2.57 13.3 11.3
Grape pomace
5
1.09 0 47.1
1
From NRC (1985).
2
Estimated values from corn gluten (NRC, 1985).
3
Calculated from NRC (1985). Rabbit pellets contain 15% barley,
7.5% wheat, 8.05% canola meal, 1.2% fat, 51.9% alfalfa, 10% wheat
bran, and 6.35% minerals and premixes (Intermountain Farmers As-
sociation, Salt Lake City, UT).
4
Manna Pro Products (Chesterfield, MO).
5
Van Soest (1994).
2573 Oro-sensorial diversity and feeding behavior
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The model diagnostics included testing for a normal
distribution of the error residuals and homogeneity of
variance. Means were analyzed using pairwise differ-
ences of least squares means.
RESULTS AND DISCUSSION
Intake and Performance
Averaged across the 60-d period, lambs receiving the
diversity treatment consumed more (P < 0.001) feed
than lambs receiving the monotonous diets (Table 3). It
has been hypothesized that generalist herbivores, such
as sheep, prefer a diversity of feeds because no single
plant species in rangelands can provide for all of the
nutrient demands of the animal (Westoby, 1974, 1978),
and because generalist herbivores are unable to detoxi-
fy large amounts of chemically similar plant secondary
metabolites (Freeland and Janzen, 1974). Nevertheless,
herbivores prefer a diversity of feeds and flavors even
when nutritional needs can be met with single feeds or
when toxins are not a concern (Early and Provenza,
1998; Atwood et al., 2001). An alternative explanation
that encompasses this observation is that preference
for a specific flavor decreases during ingestion, whereas
liking for uneaten feeds remains unchanged, a phenom-
enon known as sensory-specific satiety (Rolls, 1986).
Thus, variety reduces the rate of habituation, lead-
ing to an increase in intake relative to feed monotony
(Epstein et al., 2009). The process of ingesting a feed
induces satiety for that particular feed, motivating ani-
mals to seek alternative feeds and flavors (Provenza,
1996). Exposure, even to a nutritionally balanced feed,
can decrease preference for that feed in sheep and cat-
tle, and the decrease in preference is more pronounced
when the feed is nutritionally unbalanced (Early and
Provenza, 1998; Atwood et al., 2001).
Increases in intake caused by feed variety could be
mediated by sensory signals, postingestive signals, or
both; consequently, the mechanisms underlying such a
response could be confounded. In the current study, the
postingestive effects were controlled because the same
ration was used across treatments and nonnutritive fla-
vors were provided to create diversity. Likewise, feeding
rats a high-energy diet containing a variety of flavors
produced an increase in energy intake and BW gains
(Treit et al., 1983; Naim et al., 1986). Collectively, the
positive effects of variety on intake suggest that flavor
diversity reduces the sensory-specific satiety induced by
repeated exposure to the same flavored feed.
Lambs in the diversity treatment consumed equiva-
lent amounts of the plain and umami diets, with uma-
mi being consumed in greater amounts (P < 0.001)
than the bitter and sweet diets (Table 4). The plain
diet was the most familiar to the lambs because they
had initially been exposed to alfalfa and barley along
with their mothers. Moreover, given that the umami
receptors can sense several AA and simple peptides
that derive from the breakdown of proteins, it can be
argued that the umami taste evolved to recognize pro-
tein sources (Temussi, 2009). Growing ruminants may
display a particular preference for umami feeds because
their requirement for protein is high, and umami sig-
nals the presence of protein (Luscombe-Marsh et al.,
2008). Because growing ruminants have greater needs
for protein, it is likely that this effect enhanced intake
of the umami-flavored diet relative to the bitter- and
sweet-flavored diets. In addition, the lesser preference
for the bitter-tasting ration could be attributed to the
anorectic effects of bitterness (Chen et al., 2006). The
incapacity of the sweet diet to stimulate feed intake in
the current study differs from results described else-
where, which reported an increase in DMI in response
to feeding sweetened diets to steers (McMeniman et
al., 2006) or calves (Montoro et al., 2010). A potential
explanation for the relatively low consumption of the
sweet diet could be the fact that the sweet flavor was
not associated with an increased caloric supply from
the ration. Likewise, lambs exposed to a noncaloric
sweetener (saccharin) associated with a flavor mani-
fested decreased preferences for that flavor compared
with lambs exposed to the same flavor but paired with
calories from glucose (Burritt and Provenza, 1992).
Nevertheless, lambs still preferred the umami flavor
even when its ingestion was not associated with an
increased reward (e.g., protein supply). One possible
Table 3. Feed intake and performance of 5 groups of lambs exposed during a 60-d period to the same feed offered
in different flavors: plain, bitter, sweet, umami, and diversity (a simultaneous offer of the 4 flavored feeds)
Item
Treatment
SE
P-value
1
Plain Bitter Sweet Umami Diversity
2
Trt Time Trt time
Feed intake, g/kg
of BW
0.75
per day
110.16
c
114.28
b
110.13
c
114.25
b
119.21
a
1.22 <0.001 <0.001 <0.001
Initial BW, kg 23.0
b
22.7
b
28.5
a
26.0
a
22.2
b
1.55 0.03
Final BW, kg 38.5 38.9 43.4 42.6 40.5 2.03 0.35
ADG, kg 0.19 0.20 0.19 0.21 0.25 0.014 0.06 0.01 0.24
Feed efficiency 0.13 0.13 0.11 0.12 0.14 0.007 0.051 0.008 0.51
ac
Different superscripts within a row indicate significant differences at P 0.05.
1
Trt = effect of treatment; time = effect of time; Trt time = interaction between treatment and time.
2
A simultaneous offer of the 4 flavored feeds.
2574 Villalba et al.
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reason for this dichotomy may reside in the different
neural mechanisms by which sweet and umami tastants
signal reward. For instance, dopaminergic neurons of
the midbrain, which have been implicated in the re-
warding aspects of food, influence sucrose consumption
but do not alter the consumption of umami compounds
(Shibata et al., 2009). Additionally, it has been sug-
gested that the sweet taste produced by some mono- or
disaccharides is different from that induced by some
glucose polymers and artificial sweeteners. This differ-
ence is attributed to a differential activation of brain
dopamine, serotonin receptor systems, or both involved
in the learning of flavor preference (Bonacchi et al.,
2008).
Total feed intake of lambs in the diversity treatment
was affected by an interaction (P < 0.001) between
time and treatment (Figure 1). No differences among
treatments were observed until d 19 of the study, and
then between d 19 and 43, lambs in the diversity treat-
ment had the greatest intakes among all the treatments
(P < 0.05); thereafter, from d 44 until the end of the
period, feed consumption once again did not differ
among treatments. From d 1 to 15, lambs in the diver-
sity treatment selected primarily plain (18.4 2.5 g/
kg of BW
0.75
) and umami (21.5 2.5 g/kg of BW
0.75
)
feeds over sweet (15.0 2.5 g/kg of BW
0.75
) and bitter
(15.5 2.5 g/kg of BW
0.75
) feeds, whereas from d 19 to
43, they displayed a more even consumption of flavors:
30.9, 35.5, 34.3, and 36.1 2.5 g/kg of BW
0.75
for the
bitter, plain, sweet, and umami feeds, respectively (fla-
vor day; P < 0.001). This generalist effect during
d 19 to 43 might have contributed to an enhancement
of feed intake during that period. By d 58, however, the
numerically greater concentrations of plasmatic leptin
in the diversity treatment (Table 5) may have reduced
the differences in feed intake among treatments toward
the end of the 60-d period.
No differences in ADG were found among treatments,
although lambs in the diversity treatment tended (P =
0.06) to grow faster than lambs in the other treatments
(Table 3). Lambs in the diversity and plain treatments
also tended (P = 0.051) to show a greater feed efficien-
cy than those in the rest of the treatments (Table 3).
As discussed below, these tendencies toward improved
performance of lambs in the diversity treatment could
be linked to increases in intake and changes in the eat-
ing pattern.
Eating Pattern and Plasma Hormone
Concentrations
When feed consumption was recorded at 30-min in-
tervals during d 55, total feed intake was different (P <
0.05) among treatments across time (Figure 2). Animals
showed 2 pronounced peaks of feed intake, coinciding
with 30 and 270 min postfeeding (Figure 2). Lambs
receiving a diversity of flavors (diversity treatment)
showed less (P < 0.05) intake than those in the other
treatments during the 2 peaks of feed consumption (i.e.,
at 30 and 270 min postfeeding). However, in agreement
with the greater total daily intakes observed in the di-
versity treatment (Table 3), lambs in this treatment
consumed more (P < 0.01) feed than those exposed
to monotonous flavors at 60 (diversity > plain, bitter,
umami), 90 (diversity > plain, sweet, umami), 120 (di-
versity > plain, bitter, sweet), and 180 min (diversity >
plain, bitter, sweet, umami) after feeding (Figure 2). In
addition, lambs offered a diversity of flavors consumed
more (P < 0.05) umami (3.12 0.20 g/kg of BW
0.75
)
and plain (2.84 0.20 g/kg of BW
0.75
) feeds than sweet
(2.26 0.20 g/kg of BW
0.75
) and bitter (2.18 0.20
g/kg of BW
0.75
) feeds (data not shown). This pattern
was a consequence of differences in the intake of fla-
vored feeds observed after 30 (plain, umami > bitter,
sweet), 60 (umami > bitter), 90 (plain > bitter), 180
(plain, umami > bitter), and 270 (bitter > sweet) min
of feeding the rations (flavor time; P = 0.03). Results
from the present study suggest that lambs exposed to a
variety of flavors achieved greater intakes than animals
exposed to single rations because they consumed more
(P < 0.05) feed between the 2 peaks of consumption
(at 30 and 270 min postfeeding) than did lambs ex-
posed to monotonous diets. Alternating consumption
of flavored feeds by lambs exposed to multiple flavors
likely reduced the rate of consumption at peaks of feed
intake (i.e., when animals exposed to monotonous diets
likely manifested their greatest intake rate capacity).
The process of exercising selectivity reduces the eating
rate in ruminants (Chapman et al., 2007), an inefficien-
cy that could not have occurred in treatments exposed
to monotonous diets because they had no alternatives.
Furthermore, because the orexigenic hormone ghrelin
was reported to increase as the time that elapsed since
the last meal increased (Shintani et al., 2001), the least
plasma ghrelin concentrations observed in lambs in the
Table 4. Feed intake by a group of lambs (diversity treatment) exposed during a 60-d period to a simultaneous
offer of the same feed in different flavors: plain, bitter, sweet, or umami
1

Item
Treatment
SE
P-value
2
Plain Bitter Sweet Umami Trt Time Trt time
Feed intake, g/kg of BW
0.75
per day 31.0
b
26.6
c
28.3
b
33.3
a
0.56 <0.001 <0.001 <0.001
ac
Different superscripts within a row indicate significant differences at P 0.05.
1
A simultaneous offer of the 4 flavored feeds.
2
Trt = effect of treatment; time = effect of time; Trt time = interaction between treatment and time.
2575 Oro-sensorial diversity and feeding behavior
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diversity treatment, compared with those in the other
treatments (Table 5), could be partly responsible for
the reduced intake values at the 2 peaks of feed con-
sumption (i.e., 30 and 270 min postfeeding). On the
other hand, although values did not differ statistically,
the decreased concentrations of the anorectic peptides
CCK and GLP-1 in the plasma of lambs from the di-
versity treatment, compared with those in the other
treatments, might have caused a reduction in satiety
between peaks of consumption (i.e., 60, 90, 120, and
180 postfeeding), allowing feed intake to increase at
these times (Cummings and Overduin, 2007). In ad-
dition, the simultaneous offer of preferred (palatable)
flavors to lambs in the diversity treatment might have
enhanced the activity of the endocannabinoid system,
which, in turn, could delay the induction of satiety by
CCK (Di Marzo and Matias, 2005). Alternatively, the
increase in feed intake observed in lambs consuming
a diversity of feeds may not have represented a com-
pensation for reduced intakes at peaks of consumption,
Figure 1. Daily feed intake of lambs exposed to the same feed offered in different flavors: bitter, plain, sweet, umami, and diversity (a simul-
taneous offer of the 4 flavored feeds). Between d 19 and 43, lambs in the diversity treatment had the greatest intakes among all treatments (P <
0.05). Values are means for 7 lambs; SE are represented by vertical bars.
Table 5. Plasma hormone concentrations as affected by dietary treatments
Item
Treatment
SE
P-value
1
Plain Bitter Sweet Umami Diversity
2
Trt Time Trt time
Insulin, ng/mL 0.62 0.76 0.72 0.89 0.82 0.067 0.13 <0.001 <0.001
Leptin, ng/mL 1.48 1.26 1.51 1.46 1.53 0.072 0.14 0.13 0.46
Ghrelin, pg/L 287.0 361.0 383.8 328.8 225.9 35.50 0.06 0.17 0.71
IGF-1, g/L 74.7 90.3 80.08 97.6 100.2 7.48 0.16 <0.001 0.04
Cholecystokinin, pmol/L 9.27
a
7.96
b
7.45
bc
7.86
b
6.63
c
0.291 <0.001 <0.001 <0.001
Glucagon-like peptide 1, pg/mL 151.9
a
132.2
c
129.7
c
144.2
b
126.3
c
3.62 <0.001 <0.001 <0.001
ac
Different superscripts within a row indicate significant differences at P 0.05.
1
Trt = effect of treatment; time = effect of time; Trt time = interaction between treatment and time.
2
A simultaneous offer of the 4 flavored feeds.
2576 Villalba et al.
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but rather an enhancement in the daily consumption of
feed. Results also showed that when offering a variety
of flavors, feed intake was more evenly spread through-
out the day. Because ruminants do not eat continuously
throughout the day but eat in bouts (Burt and Dunton,
1967; Friggens et al., 1998), flavor variety may influence
daily intake through changes in the distribution of feed-
ing events over time (i.e., through changes in the feed-
ing pattern). In addition, changes in feeding frequency
may lead to physiological changes in the animal, such
as a modification in the profiles of hormones involved
in the control of feed intake (Bello et al., 2009) and
nutrient utilization (Bunting et al., 1987). Lambs in
the diversity treatment showed the greatest leptin and
IGF-1 plasma concentrations (Table 5 and Figure 3,
respectively) and lambs in the plain treatment showed
the least plasma insulin concentrations at 200 and 300
min postfeeding (Figure 3), which would indicate that
glucose supply at these times was low. The 3 hormones,
leptin, IGF-1, and insulin, have been associated with
the energy status (Chilliard et al., 2005) of the animal,
and it would be reasonable to expect that a greater in-
take would elicit an increase in plasma concentrations
of these 3 hormones.
The bitter taste reduces palatability (Grovum and
Chapman, 1988; Gherardi and Black, 1991) and in-
creases CCK release by enteroendocrine cells (Chen et
al., 2006), a hormone that suppresses intake in rumi-
nants (Baldwin, 1985). However, in the current study,
plasma CCK concentrations were greatest (P < 0.001)
for the plain diet, followed by the bitter- and umami-
flavored diets (Table 5). The bitter diet elicited the
greatest (P < 0.001) release of CCK at 60 min post-
feeding, but then it decreased below the concentrations
observed with the plain diet (Figure 3). The decreased
preference for the sweet-flavored diet was not expect-
ed because sweetness typically enhances intake and
acceptability in large ruminants (McMeniman et al.,
2006; Montoro et al., 2010).
Scan Sampling
No differences among treatments were detected in
the proportion of scans of eating events recorded during
the feeding cycle (data not shown). Thus, the differ-
ences in feed consumption observed among the differ-
ent flavors within the diversity treatment were likely
supported by changes in eating rate, but not eating
frequency. This observation is in agreement with the
notion that alterations in the amount of feed consumed
in response to feed palatability are largely mediated
by changes in the rate and frequency of eating (Davis
and Smith, 1988). It therefore seems reasonable to sug-
gest that when lambs alternated their consumption of
flavored feeds when exposed to multiple flavors, they
likely reduced the rate of consumption, particularly at
peaks of feed intake.
Figure 2. Feed intake at 30-min intervals by lambs exposed to the same feed offered in different flavors: bitter, plain, sweet, umami, and
diversity (a simultaneous offer of the 4 flavored feeds). Lambs in the diversity treatment showed the least intakes at 30 and 270 min (P < 0.05),
but they consumed more feed than lambs in the plain (at 60, 90, 120, and 180 min), bitter (at 60, 120, and 180 min), umami (at 60, 90, 180 min),
and sweet (at 90, 120, and 180 min; P < 0.01) treatments. Values are means for 7 lambs; SE are represented by vertical bars.
2577 Oro-sensorial diversity and feeding behavior
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Preferences for Novel Feeds
Few experimental studies have been conducted on
the effects of early experiences with feed on subse-
quent feed acceptance. Generalization across sensory
stimuli (e.g., flavor) seems to be an important mecha-
nism by which herbivores accept novel feeds (Augner et
al., 1998). Sheep are able to generalize in a qualitative
and quantitative fashion across cues provided by pal-
atable (Villalba and Provenza, 2000) and unpalatable
(Launchbaugh and Provenza, 1993) feeds.
No differences in intake of novel high-energy feeds
were detected among treatments (treatment novel
feed; P = 0.49; treatment novel feed day; P =
0.86; data not shown). Likewise, no differences in pref-
erences for such feeds were found among treatments
(P = 0.13). However, there was an interaction between
treatment and novel feed and day (P < 0.01; Figure 4).
For the first day of testing, lambs in the plain treat-
ment showed a greater preference for beet pulp than
did lambs fed the umami and bitter diets (Figure 4).
On d 2, lambs in the plain treatment (58 7.6%) had a
greater preference for milo than did lambs in the sweet
(23 7.6%) and diversity treatments (27 7.6%), and
lambs in the sweet treatment (39 7.6%) had a greater
preference for corn than did lambs fed the plain (12
7.6%) and bitter (18 7.6%) diets. Corn contains
a relatively high concentration of sucrose (Kuo et al.,
1988), and lambs may have generalized the sweet taste
of corn from their previous experiences with the fla-
vored ration.
No differences in total intake or preferences for novel
high-protein feeds were detected among treatments (P
> 0.10; data not shown). Similarly, no differences in in-
Figure 3. Evolution of plasma insulin (A), cholecystokinin (CCK; B), IGF-I (C), and glucagon-like peptide 1 (GLP-1; D) concentrations in
lambs exposed to the same feed offered in different flavors: bitter, diversity (a simultaneous offer of the 4 flavored feeds), plain, sweet, and umami.
Values are means for 3 lambs; SE are represented by vertical bars. Asterisks indicate significant differences (P 0.05) among treatments at each
particular time point.
2578 Villalba et al.
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takes of novel phytochemical-containing feeds were de-
tected among treatments (P > 0.10; data not shown).
However, there was a tendency (P = 0.06) for lambs
in different treatments to consume different amounts
of novel feeds over time. Specifically, during the last
day of testing, lambs in the diversity (6.9 0.92 g/
kg of BW
0.75
), plain (8.8 0.92 g/kg of BW
0.75
), and
sweet (7.1 0.92 g/kg of BW
0.75
) treatments tended to
consume more saponin-containing feed than did lambs
in the bitter treatment (4.6 0.92 g/kg of BW
0.75
),
and lambs in the bitter (5.9 0.92 g/kg of BW
0.75
)
and diversity treatments (6.3 0.92 g/kg of BW
0.75
)
Figure 4. Interactions between day, treatment, and type of novel feed on feed preferences of lambs previously exposed for 60 d to the same feed
offered in different flavors: bitter, plain, sweet, umami, and diversity (a simultaneous offer of the 4 flavored feeds). Lambs in the plain treatment
> lambs in the umami and bitter treatments [beet pulp (A); d 1; P < 0.05], and lambs in the sweet and diversity treatments [milo (B); d 2; P <
0.05]. Lambs in the plain and umami treatments > lambs in the bitter treatment [saponins (C); d 4; P < 0.10]. Lambs in the bitter treatment >
lambs in the plain and sweet treatments [tannins (D); d 4; P < 0.10]. Lambs in the plain and bitter treatments > lambs in the diversity treatment
[wheat straw (E); d 1; P < 0.05]. Lambs in the diversity treatment > lambs in the plain treatment [wheat bran (F); d 1; P < 0.05]. Treatment
novel feed day; P < 0.05. Values are means for 7 lambs; SE are represented by vertical bars.
2579 Oro-sensorial diversity and feeding behavior
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consumed more tannin-containing feed than did lambs
in the umami treatment (3.7 0.92 g/kg of BW
0.75
).
Furthermore, the evolution of feed preferences differed
among treatments and novel feeds across time (P <
0.05). During the last day of testing, lambs in the plain
and umami treatments showed a greater preference for
saponin-containing feed than did lambs in the bitter
treatment, and lambs in the bitter treatment showed a
greater preference for tannin-containing feed than did
lambs in the plain and sweet treatments (Figure 4).
Lambs in the bitter treatment may have generalized the
bitter taste of tannins from their previous experiences
with the flavored ration because tannins are bitter-tast-
ing compounds (Jackson et al., 1996).
Finally, no differences in total intake of novel low-
quality feeds were detected among treatments (data
not shown). However, for the first day of testing, lambs
in the plain and bitter treatments showed a greater (P
< 0.05) preference for wheat straw than did lambs in
the diversity treatment, whereas lambs in the diversity
treatment showed a greater (P < 0.05) preference for
wheat bran than did lambs in the plain treatment (Fig-
ure 4).
It has been shown that infants exposed for 8 d to
a variety of fruits (not including pears) manifested a
greater consumption of pears but this acceptance did
not generalize to green beans (more bitter and less
sweet than pears). In contrast, infants exposed for 8
d to a variety of vegetables tended to eat more green
beans after exposure (Mennella et al., 2008). Likewise,
infants who were exposed to different starchy vegeta-
bles (not including carrots) each day ate as many car-
rots after exposure as infants who were repeatedly ex-
posed to carrots (Gerrish and Mennella, 2001). Thus, it
seems that for the acceptance of novel feeds, the flavor
of the feeds experienced in the past and how it relates
to the target feed are important (Mennella et al., 2008).
Some of the observed preferences in the current study
are consistent with this notion. Studies of rats and hu-
mans suggest that exposure to a variety of feeds is more
robust when there are pronounced sensory differences
between feeds (Rolls et al., 1981, 1983). Flavors in the
current study were contrasting because they targeted 3
out of the 5 known primary tastes. It could be that ex-
posure to such multiple sensory contrasts may enhance
the transfer of diversity effect (Capretta et al., 1975),
such that by providing a varied flavor experience, it
would lead to a subsequent increased acceptance of
novel feeds and flavors. However, the effect of experi-
ence with or exposure to flavor diversity did not neces-
sarily lead to a clear pattern of an improved acceptance
of all the novel feeds tested.
In summary, exposure to diverse flavors, independent-
ly of postingestive events, has the potential to stimulate
intake, alter eating behavior, and improve animal per-
formance. These differences are likely attributed to a
sustained need for protein in growing animals (umami)
and the rejection of toxic compounds of plant origin
(bitter). Repeated exposure to flavored feeds influences
subsequent acceptance of novel feeds. Although this ef-
fect is subtle, and, for many feeds, short-lived, it may
have a significant effect on attenuating transient, but
economically meaningful, reductions of feed intake and
productivity during periods of transition when animals
are introduced to novel feeds. Flavor diversity has the
potential not only to enhance feed intake, but also to
induce a more even spread of feed intake throughout
the day. This effect could reduce rumen pH fluctuations
(Sutton et al., 1986), potentially enhancing the syn-
chrony of nutrient supply to the host (Hersom, 2008).
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