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from the one found in squirrel monkeys [26], spider mon- keys [30], olive baboons [32], .... tatory responsiveness to food-associated acids in the spider monkey.

Physiology & Behavior 76 (2002) 335 – 341

Gustatory responsiveness to food-associated saccharides in European rabbits, Oryctolagus cuniculus Matthias Laska* Department of Medical Psychology, University of Munich Medical School, Goethestr. 31, D-80336 Munich, Germany Received 3 January 2002; received in revised form 14 March 2002; accepted 28 March 2002

Abstract Taste preference thresholds for six food-associated saccharides and relative sweet-taste preferences were assessed in 12 European rabbits using two-bottle choice tests of brief duration (3 min). In Experiment 1, the animals were found to significantly prefer concentrations as low as 30 mM maltose, 50 mM polycose and sucrose, 75 mM glucose, 150 mM fructose, and 175 mM lactose over tap water. In Experiment 2, the rabbits were given a choice between all binary combinations of the same six saccharides presented in equimolar concentrations of 100 and 200 mM. Preferences for individual saccharides were stable across the concentrations tested and indicate the following order of relative effectiveness: maltose=polycose > sucrose>glucose>fructose lactose. The results showed rabbits to display a pattern of taste preference thresholds and relative taste preferences for food-associated saccharides similar to that found in rodents but differing markedly from that found in human and nonhuman primates. The results support the assumptions that rabbits, unlike most primates, but similar to rodents, may have specialized taste receptors for starch, and that the gustatory responsiveness of Oryctolagus cuniculus to food-associated saccharides might reflect an evolutionary adaptation to its dietary habits. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Rabbits; Oryctolagus cuniculus; Gustatory performance; Taste preference thresholds; Relative sweetness; Food-associated tastants

1. Introduction Rabbits have been extensively used in studies on gustatory neuroanatomy (e.g. [1,14,21,35,50]) and electrophysiology (e.g. [8,42 – 44,51]). Surprisingly little, however, is known about taste performance of rabbits at the behavioral level. Most studies so far have either concentrated on detectability of the four basic taste qualities, usually using sucrose, sodium chloride, hydrochloric acid, and quinine hydrochloride as the only, prototypic, stimuli [4,6,11], or have assessed the ability of rabbits to respond to artificial sweeteners [16] or taste modifiers [17]. There is only sparse information as to the taste responsiveness of rabbits for naturally occurring saccharides other than sucrose. Plants that make up the food of rabbits, however, usually contain a variety of substances termed sweet by humans [23], and the food selection behavior of rabbits suggests that they may use gustatory cues to assess palatability and nutritional value of a potential food item [20].

* Tel.: +49-89-5996-655; fax: +49-89-5996-615. E-mail address: [email protected] (M. Laska).

Sucrose, together with its monosaccharide components fructose and glucose, may account for more than 90% of total carbohydrate content in fruits consumed by rabbits [36]. Milk, as the first diet in the life of any mammal, contains considerable amounts of lactose as its almost exclusive soluble carbohydrate, ranging from 2 to 10 g/ 100 ml in herbivores [37]. Maltose, although only rarely present in plant material in free form [49], originates in considerable amounts from enzymatic degradation of starch during mastication through the salivary enzyme amylase [7] and thus enters into the complex taste sensation while feeding on starchy plants. Starch, finally, is the most abundant carbohydrate in the vegetative parts of plants [19] and thus represents a major source of metabolic energy for generalist herbivores such as rabbits. It was therefore the aim of the present study to assess the gustatory responsiveness of rabbits to these six food-associated saccharides that are all important constituents of the natural diet in Oryctolagus cuniculus [10]. For this, a twobottle preference test of brief duration was employed. This method makes it possible to determine preference thresholds—as a first and conservative approximation of the gustatory capacity of a species—as well as to directly measure relative preferences for or aversions to tastants.

0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 7 1 9 - 9

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A comparison of such basic measures of taste performance in the rabbit to those of other mammalian species also allowed me to address the question of whether the gustatory responsiveness of rabbits may mirror an evolutionary adaptation to their dietary habits.

2. Materials and methods 2.1. Animals Testing was carried out using four adult male and eight adult female European rabbits (O. cuniculus) of an established chinchilla strain (Chbb-Ch; Thomae, Biberach, Germany). The animals were kept indoors under natural light conditions at 18 – 22 C. They were housed individually, and were provided with commercial rabbit pellets (Ssniff, Soest, Germany), hay and fresh vegetables ad libitum, but were deprived of water overnight (that is, from 8 p.m. to 8 a.m.) before testing the following morning. The amount of food offered daily was such that leftovers were still present on the floor the next morning; thus, it is unlikely that ravenous appetite affected the animals’ behavior during the test. The experiments reported here comply with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, revised 1985) and also with current German laws. 2.2. Procedures In all experiments, a two-bottle choice test of brief duration was employed. Twice each day, approximately 2 and 1 h, respectively, before feeding, the animals were allowed 3 min to drink from a pair of simultaneously presented 750-ml cylinders with metal drinking tubes. In Experiment 1, the rabbits were given the choice between tap water and defined concentrations of the saccharides sucrose, glucose, fructose, lactose, maltose (reagent grade, Merck, Darmstadt, Germany), and polycose (reagent grade, Ross Products, Columbus, USA) dissolved in tap water. Polycose is a highly water-soluble mixture of glucose chains with a-1,4 linkages (2% glucose, 7% maltose, 55% maltooligosaccharides of 3 – 10 glucose units, and 36% maltopolysaccharides of >10 glucose units). It is produced from an acid enzyme hydrolysis of (unsoluble) corn starch and is frequently used as a taste stimulus in studies of gustatory performance. For each of these substances, testing started at a concentration of 400 mM and proceeded in the following steps (200, 100, 50, 20, 10 mM, etc.) until the animals failed to show a significant preference. Subsequently, intermediate concentrations were tested in order to determine the preference threshold value more exactly. To keep up the animals’ motivation and willingness to cooperate, testing did not follow a strict descending staircase procedure but followed a pseudorandomized scheme in which trials with

high and thus presumably readily perceptible and attractive concentrations of saccharides were alternated with low and thus presumably less attractive concentrations. With all animals, the order of testing was: sucrose, fructose, glucose, maltose, lactose, polycose. Retests with sucrose and maltose at the end of the study showed excellent agreement with the results of the initial test series suggesting that the order of testing did not affect the outcome. In this, as well as in the following experiment, the solutions were allowed 24 h to mutarotate. In Experiment 2, the rabbits were given the choice between all binary combinations of sucrose, fructose, glucose, lactose, maltose, and polycose presented in equimolar concentrations. In order to assess whether relative preferences are stable at different concentration levels, two test series were performed: at 100 and 200 mM, respectively. In both experiments, each pair of stimuli was presented 10 times per animal, and the position of the stimuli was randomized in order to counterbalance possible position preferences. 2.3. Data analysis For each animal, the amount of liquid consumed from each bottle was recorded, summed for the 10 test trials with a given stimulus combination, converted to percentages (relative to the total amount of liquid consumed from both bottles), and 66.7% (i.e. 2/3 of the total amount of liquid consumed) was taken as criterion of preference. This rather conservative criterion was chosen for reasons of comparability of data as the same criterion had been used in previous studies using the same method with other mammalian species [24 –34], and in order to avoid misinterpretation of data due to a too liberal criterion. Additionally, two-tailed binomial tests [48] were performed and an animal was only regarded as significantly preferring one of the two alternative stimuli if it reached the criterion of 66.7%, and consumed more from the bottle containing the preferred stimulus in at least 8 out of 10 trials (binomial test, P < .05). Preliminary analysis of the data indicated that there were no reliable differences in choice behavior and liquid consumption between the male and the female subjects and between the first and the second presentation of the day. Intraindividual variability in the amount of liquid consumed across the 10 test trials with a given stimulus combination was low and averaged less than 20%. Thus, a theoretically possible bias in the overall preference score due to excessive drinking in aberrant trials did not occur. Therefore, the data for the male and the females obtained in the 10 test trials were combined and are reported as group means and standard deviations. Comparisons across tasks were made using the Friedman two-way analysis of variance (ANOVA). When ANOVA detected differences between tasks, this was then followed by separate pairwise Wilcoxon signed-rank tests for related samples to evaluate which tasks were involved.

M. Laska / Physiology & Behavior 76 (2002) 335–341

3. Experiment 1: Taste preference thresholds for food-associated saccharides 3.1. Results Fig. 1 shows the taste preference thresholds of O. cuniculus to be 30 mM for maltose, 50 mM for polycose and sucrose, 75 mM for glucose, 150 mM for fructose, and 175 mM for lactose. All rabbits significantly discriminated these concentrations from tap water, and in some cases, single individuals even scored slightly lower preference threshold values. All animals, however, failed to show a

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significant preference for the lowest concentrations presented, suggesting that the preference for higher concentrations was indeed based on the chemical nature of the stimuli. In most cases, interindividual variability of scores was low for both sub- and suprathreshold concentrations tested (cf. S.D.s in Fig. 1). Suprathreshold concentrations of all six saccharides tested were highly preferred over tap water and led to marked concentration-dependent polydipsia, i.e. consumption of liquid in excess of physiological need. The mean total volume of liquid consumed per trial and animal already increased significantly (Wilcoxon, P < .05) with the first

Fig. 1. Taste responsiveness of six rabbits to aqueous solutions of sucrose, maltose, glucose, fructose, lactose, and polycose tested against tap water. Each data point represents the mean value (± S.D.) of 10 test trials of 3 min per animal.

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M. Laska / Physiology & Behavior 76 (2002) 335–341

suprathreshold concentration compared to baseline intake with subthreshold concentrations, and increased up to between two- and fourfold in the presence of 200 or 400 mM solutions of the individual saccharides tested. 3.2. Discussion Table 1 compares the taste preference threshold values obtained in the present study with those obtained with the same or a similar method in other mammalian species for which data from at least three of the six substances tested are at hand, and with detection threshold values obtained for humans, using psychophysical procedures. For all six saccharides tested, O. cuniculus responded to slightly or even moderately higher concentrations compared to most other mammalian species investigated so far, and

Table 1 Taste preference thresholds (mM) for food-associated saccharides in rabbits and other mammalian species Species

Sucrose Fructose Glucose Lactose Maltose Polycose

Oryctolagus 50 cuniculusa Homo 10 sapiensb,c Ateles 3 geoffroyid,e Saimiri 10 sciureusd,f Papio hamadryas 10 anubisd,g Macaca 10 nemestrinad,h Cebuella 33 pygmaeai Saguinus midas 66 nigeri Rattus 5 norvegicusj,k,l Meriones 5 unguiculatusj Mesocricetus >10 auratusj Acomys >10 cahirinusj

150

75

175

30

50

40

80

72

38

30

15

20

10

20

30

40

90

100

90

60

20

25

20

20

30

20

20

30

10

10

50

100

125

66

330

>250

40

10

133

5

10

5

>10

sucrose polycose =m altose>sucrose

Homo sapiensb 100 200

sucrose>maltose = polycose sucrose>maltose>polycose

Saimiri sciureusc 100 200

sucrose>polycosemaltose sucrose>polycose = maltose

Ateles geoffroyic 100 200

sucrose>polycose  maltose sucrosemaltose  polycose

Papio hamadryas anubisc 100 200

sucrose>polycose>maltose sucrose>polycose  maltose

Macaca nemestrinac 100 200

maltose>polycose  sucrose maltose = polycose = sucrose

Rattus norvegicusd 30 100 300

polycose>maltose>sucrose polycose  maltose  sucrose polycose  sucrose  maltose

The study in Ref. [9] established relative sweetness rather than preference. a Present study. b Ref. [9]. c Ref. [32]. d Ref. [46].

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M. Laska / Physiology & Behavior 76 (2002) 335–341

receptors may not be restricted to rodents but may also be found in members of other mammalian orders such as primates or—as in the present study—in lagomorphs.

5. General discussion The present study sought to assess the gustatory responsiveness of European rabbits to food-associated saccharides. Two main findings emerged from the two-alternative choice tests employed. First, rabbits generally responded to slightly or even moderately higher concentrations compared to most other mammalian species investigated so far, and thus, do not appear to rank among the most saccharide-sensitive mammals. Second, rabbits displayed a pattern of taste preference thresholds and relative taste preferences for food-associated saccharides which is similar to that found in rodents but differs markedly from that found in most primates. The question arises as to possible reasons that might account for the observed pattern of taste responsiveness. Differences in dietary habits have been shown repeatedly to provide plausible explanations for differences in taste performance among species [22,38]. Among New World primates, for example, the degree of frugivory has been found to correlate positively with sensitivity to food-associated mono- and disaccharides [28]. This seems to make sense in terms of optimal foraging theory, as frugivorous species rely on soluble carbohydrates to meet their energy requirements more than nonfrugivores [34]. Sclafani [45] proposed that the high sensitivity and preference for polycose and other starch-derived polysaccharides as well as for maltose observed in the rat may also be explained by its dietary habits. Anatomical features such as teeth and gut morphology suggest that members of the genus Rattus are primarily granivorous, i.e. they feed on a starch-rich diet, lending support to this hypothesis. The taste preference thresholds of rabbits for maltose and polycose found here—although higher than those found in rodents—suggest that these saccharides are very likely to add to the taste sensation while feeding on starchy plants. As a considerable proportion of the plants that are known to be part of this species’ diet in the wild [5,10] contains high amounts of starch, it seems reasonable to assume that both the sensitivity of O. cuniculus for polycose and maltose and its marked preference for this polysaccharide and its disaccharide constituent may represent an evolutionary adaptation of the gustatory system of the rabbit to the nutritional assessment of starchy plants. This supposition is supported by the fact that rabbits have been shown to excrete extraordinarily high amounts of amylase—an enzyme dissociating starch into maltose units—in the saliva produced by their parotid glands in response to stimulation with tastants termed sweet by humans [12]. The taste preference thresholds of rabbits for sucrose and glucose suggest that these two saccharides may also enter into the complex taste sensation while feeding on fruits [36].

However, the proportion of fruits in the diet of rabbits is generally low [10] and the few species of mono- and disaccharide-containing fruits consumed by rabbits are, in contrast to starch-rich vegetative parts of plants, only seasonally available. The low degree of frugivory observed in rabbits may therefore explain their comparatively low responsiveness to fructose and the lower attractiveness of sucrose and its monosaccharide constituents fructose and glucose compared to polycose and maltose. Oftedal and Iverson [37] reported the lactose content of the milk of rabbits to range from 1.8 to 2.1 g/100 ml, corresponding to a concentration of 50 –58 mM, which according to the findings of this study is clearly below the taste preference threshold and is thus unlikely to be perceptible for O. cuniculus. It is interesting to note that the milk of primates, which are known to have a ‘sweet tooth’ [38], contains between 4 and 10 g/100 ml [37], and thus concentrations of lactose are two- to fivefold higher compared to that found in the rabbit. Taken together, the results of the present study support the assumptions that rabbits, unlike most primates, but similar to rodents, may have specialized taste receptors for starch, and that the gustatory responsiveness of O. cuniculus to food-associated saccharides might reflect an evolutionary adaptation to its dietary habits.

Acknowledgments I would like to thank Artour Stepanian and Stefan Ja¨dicke for help in collecting data, and the Deutsche Forschungsgemeinschaft for financial support (La 635/10-1).

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