Ž .
and pomace was preferred to pomace–starch during day 9 to 15 P - 0.05 . This pattern quickly reversed — pomace–starch became preferred to pomace — when the foods were offered for 8
Ž .
hrday for the next 6 days P - 0.001 . These findings suggest that energy deprivation and the amount of food ingested both affected how quickly lambs discriminated between foods that
differed in energy, and that lambs needed to eat a threshold amount of an energy-rich novel food before they acquired a preference for that food. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Preferences; Starch; Energy; Supplementation; Experience; Lambs
1. Introduction
Energy supplementation can increase or decrease forage intake, depending on the Ž
kind and amount of supplement consumed and the availability of nitrogen reviewed by .
Ž Caton and Dhuyvetter, 1997 . Low levels of grain supplementation Henning et al.,
. Ž
. 1980 , or low doses of starch infused into the rumen Villalba and Provenza, 1997a ,
increase roughage intake by sheep. Supplementation with energy increases byproducts of Ž
. fermentation like volatile fatty acids VFA , which at low doses condition preferences in
Ž .
sheep Villalba and Provenza, 1996, 1997b . Likewise, sheep prefer poor-quality foods, or non-nutritive flavors in solutions, associated with intraruminal infusions of glucose
Ž .
Ž Burritt and Provenza, 1992; Ralphs et al., 1995 or starch Villalba and Provenza,
. 1997a, 1999 .
Given that byproducts of energy fermentation condition preferences, it is conceivable that ruminants may acquire preferences for poor-quality foods like straw, if straw
ingestion coincides with increased VFA production due to eating an energy-rich supplement just prior to eating the straw. If the effects of VFA from grain supplementa-
tion condition preferences for poor-quality foods, strategic supplementation could en- hance preference for plant species of lesser nutritional quality. On the other hand,
ruminants may quickly discriminate the flavor-post-ingestive effects of the supplement from the poor-quality food, in which case they would not acquire a preference for the
poor-quality food.
Our objective was to better understand how lambs discriminate among novel foods based on flavor and post-ingestive effects, and to determine if energy supplementation
affected the preferences of lambs for a poorly nutritious food. We first determined how temporal order of food ingestion and post-ingestive feedback affected preference when
Ž .
lambs were fed a poorly nutritious food straw immediately after eating an energy-rich Ž
. food milo , and when lambs were fed straw after an infusion of milo into the rumen.
We then determined how lambs discriminated which of two novel foods was highest in energy.
2. Materials and methods
All experiments were conducted at the Green Canyon Ecology Center, located at Ž
Utah State University in Logan. During each experiment, lambs Finn–Polypay–Suffolk
. crossbreds of both sexes were individually penned and had free access to mineral
Ž .
blocks and fresh water. Alfalfa Medicago satiÕa pellets were the basal diet in all
experiments. 2.1. Experiment 1
Ž . The objective of this experiment was to determine 1 if lambs preferred flavored
Ž .
wheat Triticum aestiÕum
straw, a poorly nutritious food, when its consumption Ž
. followed ingestion or intraruminal infusions of milo Sorghum bicolor subglabrescens ,
Ž . an energy-rich supplement, and 2 if previous experience with these foods affected
preference. We conducted four trials. The same animals and flavor–nutrient associations were maintained during trials 1 to 3. A new set of animals was used in Trial 4.
2.1.1. Trial 1 The objective of this trial was to determine if lambs acquired a preference for
flavored wheat straw eaten immediately after they ate milo and if familiarity with these foods affected preference.
2.1.1.1. Initial exposure to straw and milo. Straw and milo were novel foods for all lambs, and our objective was to maintain this condition as much as possible, but at the
same time increase the likelihood that all lambs would consume some straw and milo Ž
. during conditioning. Each day at 0800 h, lambs 28 kg BW were offered 25 g of straw
Ž .
1–2 cm particle size . At 0900 h, orts were collected and weighed. When a lamb consumed G 20 g of straw on 1 day, exposure to straw ended for that lamb. Exposure
also ended when the cumulative intake of straw was G 20 g for a lamb. After 9 days, all Ž
. Ž
. lambs had reached the first n s 10 or the second n s 14 criterion. On average, lambs
Ž .
ate a cumulative amount of 31 g of straw SEM s 1 throughout the 9-days period. After exposure to straw, lambs had access to alfalfa pellets from 1200 h to 1700 h. At
Ž .
1500 h, lambs were offered 100 g of milo whole grain . At 1600 h, orts were collected and weighed. When a lamb consumed G 50 g of milo 1 day, exposure to milo ended for
that lamb. Exposure also ended when the cumulative intake of milo was G 50 g. After 6 Ž
. Ž
. days, all lambs had reached the first
n s 16 or the second n s 8 criterion. On
Ž .
average, lambs consumed a cumulative amount of 82 g of milo SEM s 1 during the 6-days period. Five lambs consumed all 100 g of milo offered in 1 day.
After initial exposure to straw and milo, lambs were randomly divided in two groups Ž
. Ž
. 12 lambsrgroup . Lambs in Group 1 naive group received no more exposure to straw
Ž .
or milo until conditioning, whereas lambs in Group 2 experienced group received more Ž
. exposure to straw and milo described below . During initial exposure, cumulative
Ž .
intake for lambs in Groups 1 and 2 was 29 g and 34 g SEM s 1 of straw, and 84 g and Ž
. 81 g SEM s 2 of milo, respectively.
2.1.1.2. Familiarization with straw and milo. From 0800 h to 1000 h, lambs in Group 2 were offered coconut-flavored straw on even days and onion-flavored straw on odd
Ž .
Ž .
days. Coconut Agrimerica, IL and onion Pacific Foods, WA flavors were mixed with
Ž .
straw at a concentration of 2 wrw . Every day at 1500 h, lambs in Group 2 were offered 200 g of milo; orts were collected at 1700. Exposure to flavored straw and milo
Ž .
lasted for 8 days. Lambs consumed 35 grday SEM s 4 of coconut-flavored straw, 35 Ž
. Ž
. grday SEM s 4 of onion-flavored straw, and 136 grday SEM s 8 of milo. All
lambs had access to alfalfa pellets from 1200 h to 1700 h. 2.1.1.3. Initial preference test. After the familiarization period, lambs in Groups 1 and 2
received coconut- and onion-flavored straw simultaneously for 15 min, and intake and preference for each flavor were determined. After the preference test, lambs in each
group were sorted in decreasing order by preference for coconut, and pairs of lambs Ž
. from high to low preference
were randomly assigned to receive that flavor in association with milo during conditioning. Thus, differences due to initial flavor
preferences were balanced. 2.1.1.4. Conditioning. On even days at 0800 h, all lambs had access to 200 g of milo for
Ž .
5 min Table 1 . Immediately after milo ingestion, half of the lambs in each group were offered coconut-flavored straw and the other half were offered onion-flavored straw for
Ž .
30 min Treatment 1 . All lambs had alfalfa pellets from 1200 h to 1700 h; no food was offered until the next day. On odd days, straw was offered to all animals but the flavors
Ž .
were switched so that lambs that received coconut- onion- flavored straw on even days Ž
. received onion- coconut- flavored straw on odd days. During odd days, no milo was
Ž .
Ž fed before straw consumption
Treatment 2 . Instead, 200 g of barley Hordeum
. Õ
ulgare grain was fed at 1600 h for 5 min. The amount of straw fed on even days was equal to a lamb’s intake of straw the
previous day, which controlled for amount of exposure to the flavored straw. Lambs in Ž
. Ž
. Groups 1 and 2 consumed, respectively, 26 SEM s 3 and 22 grday SEM s 2 of
Ž .
Ž .
flavored straw, and 188 SEM s 4 and 176 grday SEM s 7 of milo during even Ž
. Ž
. days; they ate 28 grday SEM s 3 and 21 grday SEM s 3 of flavored straw during
odd days. Ž
. 2.1.1.5. Preference test. After 8 days of conditioning four even and four odd , lambs
Ž .
were offered coconut- and onion-flavored straw simultaneously for 15 min Table 1 . Milo was not provided before the preference test. We determined intake of each food.
2.1.2. Trial 2 Trial 2 determined if restricting the supply of energy from the basal diet increased
preference for flavored straw eaten after milo consumption. Conditioning was the same as for Trial 1, but alfalfa pellets were restricted to 1100 g lamb
y1
day
y1
, which supplied Ž
. about 80 of their digestible energy requirements NRC, 1985 . Barley was not offered
Ž .
on odd days. Lambs in Groups 1 and 2 ate 62 grday and 52 grday SEM s 4 of flavored straw, and all 200 g of milo on even days. They ate 58 grday and 49 grday
Ž .
SEM s 3 of flavored straw on odd days. Lambs were tested for preference as in Trial 1. On the following day, all lambs
received coconut- and onion-flavored straw as well as milo simultaneously for 5 min. Ž
. Intake of each food was determined Table 1 .
Table 1 Methods and procedures in Experiment 1
Group Experience with straw and milo
before conditioning Ž
. Ž
. Ž
. Trials 1, 2, 3 same animals
1 Naive Limited G 20 g straw; G 50 g milo
Ž .
2 Experienced 8 days of exposure to flavored straw and
Ž .
milo 2 hrday Ž
. Trial 4
1 Naive None
Ž .
2 Experienced 8 days of exposure to flavored straw and
Ž .
milo 2 hrday
a
Trial 1 Ž
. Conditioning 8 days
Ž .
Even-numbered days: Milo followed by flavored straw Flavor 1
Ž .
Odd-numbered days: Flavored straw only Flavor 2
Preference test Ž
. Choice of flavored straw Flavors 1 and 2
b
Trial 2 Ž
. Conditioning 8 days
Same as described for Trial 1 Preference test
Ž .
Day 1: Choice of flavored straw Flavors 1 and 2 Ž
. Day 2: Choice of milo and flavored straw Flavors 1 and 2
b
Trial 3 Ž
. Conditioning 8 days
Ž .
Even-numbered days: Intraruminal infusions of milo followed by flavored straw Flavor 1 Ž
. Odd-numbered days: Flavored straw only Flavor 2
Preference test Ž
. Day 1: Choice of flavored straw Flavors 1 and 2
Ž .
Day 2: Choice of milo and flavored straw Flavors 1 and 2
b
Trial 4 Ž
. Conditioning 8 days
Same as described for Trial 1 Preference test: Milo–LiCl and Control
Ž .
Choice of flavored straw Flavors 1 and 2
a
Ž .
Basal diets: Free access to alfalfa pellets 5 hrday , and 200 g barley grain during odd-numbered days.
b
Alfalfa pellets restricted to supply 80 digestible energy lamb
y1
day
y1
.
2.1.3. Trial 3 The objective of Trial 3 was to determine lambs’ preference for flavored straw eaten
after an infusion of milo into the rumen, rather than after eating milo. Conditioning was the same as for Trial 2, but lambs were given intraruminal infusions of milo by oral
Ž .
intubation rather than fed milo Table 1 . The milo suspension was prepared by grinding milo through a 0.5-mm screen and then mixing it with tap water at room temperature.
To prevent sudden changes in the rumen environment, the level of milo infused Ž
. Ž
. increased from 100 g first infusion to 150 g second infusion and reached 200 g
during the last two infusions. Lambs in Groups 1 and 2 consumed, respectively, 82 Ž
. Ž
. grday SEM s 3 and 73 grday SEM s 3 of flavored straw on even days, and 77
Ž .
Ž .
grday SEM s 4 and 68 grday SEM s 5 on odd days. Lambs were tested for Ž
. preference as in Trial 2 Table 1 .
2.1.3.1. Effects of milo on alfalfa intake. To determine if milo infusions affected subsequent food intake, all lambs were offered the following at 0800 h for 3 days: Day 1
Ž .
— Milo 200 g and 5 min later free access to alfalfa pellets for 1 h; Day 2 — Free Ž
. access to alfalfa for 1 h; Day 3 — Milo 200 g by oral intubation and 5 min later free
access to alfalfa pellets for 1 h. 2.1.4. Trial 4
The objective of Trial 4 was to determine if the complete lack of experience with flavored straw and milo affected the acquisition of a preference for flavored straw when
straw ingestion immediately followed milo ingestion. Ž
. A new group of 24 lambs 31 kg BW was presented on day 1 with milo and straw in
temporal association as described in the Conditioning section of Trial 1. Based on day 1 intake, and a criterion of ingesting more than 5 g of milo and straw, 12 lambs were
Ž .
placed in Group 1 naive group . The rest of the lambs were placed in Group 2 Ž
. Ž
. experienced lambs . On day 1 of exposure, lambs in Group 1 ate 45 g SEM s 11 of
Ž .
Ž .
milo and 25 g SEM s 7 of flavored straw. Lambs in Group 2 ate 10 g SEM s 2 of Ž
. milo and 8 g SEM s 3 of flavored straw.
After day 1, lambs in Group 1 received no additional exposure to straw or milo. Lambs in Group 2 received more extensive experience with coconut- and onion-flavored
straw and milo, as described for Trial 1. During this familiarization period, lambs in Ž
. Ž
. Group 2 ate 23 grday SEM s 4 of coconut-flavored straw, 16 grday SEM s 2 of
Ž .
onion-flavored straw, and 188 grday SEM s 4 of milo. After familiarization, lambs in Group 2 were tested for preference and assigned to receive coconut-flavored straw in
association with milo as described for Trial 1. Ž
. Lambs in Groups 1 and 2 were conditioned as described for Trial 2 Table 1 . To be
consistent with Trial 3, milo was offered in ground form in amounts similar to those infused on successive even days in Trial 3: 100, 150, 200, and 200 g. Lambs in Groups
Ž .
Ž .
1 and 2 consumed, respectively, 42 SEM s 5 and 28 grday SEM s 5 of flavored Ž
. Ž
. straw on even days, and 33 grday SEM s 5 and 37 grday SEM s 6 on odd days.
Preference tests were conducted as described for Trial 1. 2.2. Experiment 2
The objective of this experiment was to determine if lambs discriminated between the post-ingestive effects of novel foods of different energy densities when the foods were
offered simultaneously, and if they generalized preferences based on past experiences with the oral and post-ingestive effects of starch-containing foods. Lambs in all trials
received alfalfa pellets at 1200 h as their basal diet to provide about 80 of their daily
Ž .
digestible energy requirements NRC, 1985 .
2.2.1. Trial 1 Ž
. 2.2.1.1. Exposure to noÕel foods and preference tests. Lambs
32 kg BW from
Ž .
Experiment 1 Trials 1–3 were randomly divided into two groups. The test foods and flavors used in Experiment 2 were different from those used in Experiment 1 and we
applied the same level of food restriction in both experiments. Thus, carryover effects from Experiment 1 were minimized.
Ž . At 0800 h, all lambs were offered two novel foods simultaneously: 1 a 70–30
Ž .
Ž wrw mixture of grape pomace — a low-quality food 4.6 MJrkg DE; 1.6 DP;
. Ž .
NRC, 1985 — and starch; and 2 a 70–30 mixture of grape pomace and a-cellulose. Ž
. Different flavors Apple and Maple; Agrimerica were added to the foods at a concentra-
Ž .
tion of 2 wrw to facilitate discrimination. Group 1 received the maple-flavored pomace–starch mixture and the apple-flavored pomace–cellulose mixture. Group 2
received the opposite flavorrnutrient associations. Ž
. On the first day test 1 , the foods were offered for 5 min. During the next 3 days
Ž .
tests 2 to 4 , exposure was extended to 20 min. The aim of the 5 min exposure was to determine if lambs discriminated quickly between the two novel foods. Refusals of the
Ž .
foods were collected and intake of each food was calculated Table 2 . 2.2.1.2. Exposure to the cellulose–pomace mix and preference tests. Preference for the
pomace–starch mix was nearly absolute, and thus after test 4, all lambs were exposed for 20 minrday for 2 days to only the pomace–cellulose mix in order to compensate for
the increased experience they were having with the pomace–starch mix. Lambs con-
Ž .
sumed an average of 49 g SEM s 8 of the pomace–cellulose mix. After exposure to pomace–cellulose, all lambs were again offered a choice of the two
Ž .
foods test 5 . Again, preference for the pomace–starch mix was nearly absolute, so lambs were exposed to the pomace–cellulose mix for another 20 minrday for 4 days.
Ž .
Lambs ate an average of 51 g SEM s 6 of the pomace–cellulose mix. After this Ž
. exposure, the cumulative average intake of the pomace–starch mix 355 g was similar
Ž .
to the cumulative average intake of the pomace–cellulose mix 329 g , and lambs were Ž
. again offered a choice of the two foods test 6; Table 2 .
2.2.2. Trial 2 The objective of this trial was to determine if lambs generalized an aversion from a
Ž .
Ž .
familiar grain milo to a novel food pomace that contained starch. All lambs in Trial 1 preferred pomace–starch over pomace–cellulose from the first preference test. We
hypothesized the preference for pomace–starch could be due to lambs generalizing from Ž
. familiar foods like grains, which are high in starch about 80 , to novel foods that
contain starch. After test 6, lambs in each group were sorted in decreasing order of preference for
pomace–starch. Pairs of lambs were then randomly assigned to two new groups, LiCl–Milo and Control, to balance preference for the pomace–starch and pomace–cel-
lulose between groups. The day after test 6, lambs in the LiCl–Milo group were offered milo from 0800 h
Ž .
Ž .
until 0815 h day 1 of this trial . After eating milo average intake was 430 g, SEM s 4 ,
Table 2 Methods and procedures in Experiment 2
b
Trial 1 Novel foods
Group 1 Group 2
grape pomace– qmaple flavor
qapple flavor Ž
. starch 70–30
grape pomace– a- qapple flavor
qmaple flavor Ž
. cellulose 70–30
Days Event
1–4; 7; 12 Choice of flavored grape pomace–starch and
flavored grape pomace– a-cellulose 5 minr Ž
. Ž
. day day 1 ; 20 minrday days 2–4; 7; 12 .
5–6; 8–11 Exposure to flavored pomace– a-cellulose
Ž .
without choice 20 minrday .
a
Trial 2 New groups from Trial 1
Milo ingestion before LiCl LiCl infusions
LiCl–Milo group YES
YES Control
NO YES
Ž .
Day Event after LiCl infusions
1 LiCl–Milo group receives Milo
2 Choice of flavored grape pomace–starch
Ž .
70–30 and flavored grape Ž
. pomace– a-cellulose 70–30
3 LiCl–Milo and Control
groups receive Milo 4
Choice of flavored grape Ž
. pomace–starch 30–70 and flavored
Ž .
grape pomace– a-cellulose 70–30
b
Trial 3 Ž
. Conditioning 8 days
Ž .
Even-numbered days: infusions of a-celluloseqflavored grape pomace Flavor 1 Ž
. Odd-numbered days: flavored grape pomace only Flavor 2
Preference test Ž
. Choice of flavored grape pomace Flavors 1 and 2
b
Trial 4 Ž
. Ž
. Novel foods: grape pomace–starch 70–30 and grape pomace 100
Period 1 Ž
. Days 1–15
Choice of grape pomace–starch and grape pomace 20 minrday Period 2
Ž .
Days 1–6 Choice of grape pomace–starch and grape pomace 8 hrday
Ž .
Day 7 Choice of grape pomace–starch and grape pomace 20 minrday
Ž .
Table 2 continued
a
Trial 5 New Groups from Trial 4
Milo ingestion before LiCl LiCl infusions
LiCl–Milo group YES
YES Control
NO YES
Ž .
Day Event after LiCl infusions
1 LiCl–Milo group receives Milo
2 Choice of pomace–starch and grape pomace
3 LiCl–Milo and Control Groups receive Milo
a
Ž .
Basal diets: Free access to alfalfa pellets 5 hrday .
b
Alfalfa pellets restricted to supply 80 digestible energy lamb
y1
day
y1
.
lambs in the LiCl–Milo and Control groups received by oral intubation 200 ml of a solution containing 300 mgrkg BW of LiCl, a dose that causes strong food aversions in
Ž .
sheep duToit et al., 1991 . On day 2, lambs in the LiCl–Milo group were fed milo from 0800 h to 0815 h, but no lambs ate.
On day 3, we determined if lambs generalized the aversion from milo to pomace– Ž
. Ž
. starch by measuring intake of pomace–starch 70–30 and pomace–cellulose 70–30
from 0800 h to 0815 h. On day 4, lambs were offered milo from 0800 h to 0815 h to Ž
Ž .
determine if the aversion to milo persisted milo intake was 3 g SEM s 1 in the Ž
. .
LiCl–Milo group and 421 g SEM s 13 in the Control group . On day 5, we altered the pomace–starch mix to 30–70, and offered lambs a choice between the pomace–starch
Ž .
and pomace–cellulose from 0800 h to 0815 h Table 2 . 2.2.3. Trial 3
Lambs displayed low preferences for the pomace–cellulose mix throughout Trial 1. The objective of Trial 3 was to determine if this was due mainly to oral or post-ingestive
effects. 2.2.3.1. Initial preference test. We measured intake of forage- and anise-flavored grape
Ž .
pomace offered simultaneously to 24 lambs 32 kg BW for 20 min. The flavors Ž
. Agrimerica , which were novel for the lambs, were mixed with pomace at a concentra-
Ž .
tion of 2 wrw . Lambs were sorted in decreasing order by initial preference for anise, Ž
. and pairs of lambs were randomly assigned to two groups 12 lambsrgroup so that the
groups were balanced according to initial flavor preference. 2.2.3.2. Conditioning. Lambs were conditioned during 20 minrday using the same
conditioning paradigm described in Experiment 1. Flavored grape pomace replaced flavored straw and cellulose replaced milo in the infusions. Cellulose infusions were
given immediately after lambs began to eat flavored grape pomace. The cellulose suspension was prepared mixing 250 ml tap water and 20 g of cellulose. This amount of
cellulose was the maximum amount of the pomace–cellulose mix that lambs ingested
Ž .
during a 20 min exposure in 1 day without a choice Trial 1 . Lambs consumed 155
Ž .
grday SEM s 5 when flavored pomace was not paired with cellulose, and 163 grday Ž
. SEM s 5 when flavored pomace was paired with cellulose.
Lambs were tested for preference during 20 min, as described for Experiment 1 Ž
. Table 2 .
2.2.4. Trial 4 The objective of this trial was to determine if duration of exposure influenced
Ž preference for grape pomace–starch relative to grape pomace. From days 1 to 15 Period
. Ž
. Ž
. 1 , lambs 35 kg BW from Experiment 1 Trial 4 were offered two novel foods — a
70–30 mixture of grape pomace–starch and grape pomace — simultaneously from 0800 h to 0820 h. Refusals were collected and intake of each food was calculated.
Ž .
From days 16 to 21 Period 2 , lambs were offered 500 g of each food described Ž
above, but the amount of exposure was increased to from 20 minrday to 8 hrday 0800 .
h to 1600 h daily . At 1600 h, refusals were collected and weighed and lambs received 1500 g of alfalfa pellets.
On day 22, lambs were offer both foods for only 20 min. Intake of each food was Ž
. recorded and compared with the last day of Period 1 Table 2 .
2.2.5. Trial 5 The objective of this trial was to determine if lambs generalized an aversion from a
grain high in starch to another food that contained starch. After Trial 4, lambs were Ž
. treated with LiCl as described for Trial 2; lambs consumed 372 g SEM s 51 of milo
prior to receiving LiCl, and no milo following the LiCl infusion. After LiCl infusions, Ž
. lambs in the LiCl–Milo group did not eat milo, whereas Controls ate 363 g SEM s 32 .
3. Statistical analyses
