Misidentification by wild rabbits,Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion : III. Experiments with mixed sex groups and analysis of further data from all-male and all-female groups.

The experiments described in this paper are part of a series designed to clarify the behavioral function of the odor of the secretion from the inguinal glands of rabbits,Oryctolagus cuniculus. Results are presented of tests carried out on 48 mixed-sex groups consisting of 162 adult wild-type rabbits, 48 males and 114 females. The subordinate females of the groups were smeared with various odorous materials and the reactions of their pen-mates towards them recorded. Eight sources of natural rabbit odors and a commercial perfume were used in 198 tests. A high proportion (60%) of the subordinate females smeared with the inguinal gland secretions from unfamiliar males were attacked, mainly by the males, which were generally the most dominant individuals within the groups. The paper also presents statistical analyses of behavioral data collected during two earlier related experiments with single-sex groups involving 258 rabbits in 300 tests which have not previously been evaluated. The results throughout the whole study indicate clearly that the odor of the inguinal gland secretion of rabbits carries information which affects the acceptance of individuals by their companions in organized groups. Other sources of unfamiliar rabbit odor did not influence the attitude of groupmates toward the individuals smeared with them, or if they did, their effects were only marginal.

Microflora and volatile fatty acids present in inguinal pouches of the wild rabbit,Oryctolagus cuniculus, in Australia.

Contents of inguinal pouches of the wild rabbit,Oryctolagus cuniculus, have been examined for the presence of microorganisms and volatile fatty acids. A total of 245 isolates were made from 167 samples taken from 75 rabbits, and microorganisms were obtained from 93% of individuals. The most common microorganism encountered wasStaphylococcus aureus, followed by the yeastCandida kruzei. Other organisms isolated wereBacillus subtilis,Escherichia coli, andStreptococcus faecalis. Quantitative analysis of volatile fatty acids in scrapings from pouches showed considerable variation between individuals. Of the acids identified, acetic acid and isovaleric acid were the most abundant. In a liquid medium containing one of these acids, the growth of microorganisms was only slightly affected. However, growth became inhibited when these fatty acids were used in combination.

Misidentification by wild rabbits,Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion : I. Experiments with all-male groups.

To demonstrate that the inguinal gland odor conveys information on the identity of individuals, 60 male, wild-type rabbits,Oryctolagus cuniculus, living in groups of three in outdoor pens, were used in 120 tests. Members of the groups smeared with the inguinal gland secretion from unfamiliar rabbits were attacked by their penmates. In contrast the odors of chin gland secretions and urine of strange rabbits and of a commercial perfume did not cause serious disruption to the social relationships within the groups.

Misidentification by wild rabbits,Oryctolagus cuniculus, of group members carrying the odor of foreign inguinal gland secretion : II. Experiments with all-female groups.

The results of experiments designed to clarify the behavioral function of the odor of the secretion from the inguinal glands of rabbits,Oryctolagus cuniculus, are presented. Members of groups of female rabbits were smeared with odoriferous materials, and the behavior of their penmates towards them was recorded. Eight sources of natural rabbit odors and a commercial perfume were used in 180 tests involving 120 adult female wild-type rabbits. A high proportion of the animals smeared with the inguinal gland secretions from unfamiliar females were attacked by their pen-mates. In contrast, treatment with inguinal gland secretions from unfamiliar male rabbits; macerated inguinal gland materials from unfamiliar males and females, respectively; urine from unfamiliar females; and macerated anal gland material from unfamiliar females resulted in only minor disruption to the normal social relationships between the group members which was not significantly different to that caused by treatment with a commercial perfume.

Effect of volatiles collected above fecal pellets on behavior of the rabbit,Oryctolagus cuniculus, tested in an experimental chamber : I. Total volatiles and some chemically prepared fractions.

The fecal pellets of rabbits,Oryctolagus cuniculus, are coated with secretion from the anal gland, the odor from which functions as a territorial marker. The total volatiles collected from above fecal pellets and three different fractions prepared by washing the volatiles through either distilled water, 1 M sodium hydroxide, or 1 M hydrochloric acid were tested for their effect on the territorial confidence of individual animals. Tests were based on the neutral pen paradigm used in previous studies but employed a specially designed test chamber, the internal odor environment of which could be fully controlled. In the four series of bioassays a total of 140 separate tests were performed and 64 adult male rabbits were used. The results showed that the presence of the total volatiles influences the outcome of territorial competitions in pairs of male rabbits in favor of the individual from which they were derived. They were comparable with those obtained in earlier studies when fecal pellets were presented in the test pen. The effects of the different washing reagents on the total odor are illustrated by gas chromatograms, and the components most likely to be removed by the different treatments are suggested. The volatiles remaining after passing through distilled water had less effect on the rabbit's behavior than the total odor. Since the washing process not only removed water-soluble compounds but also led to a reduction in the concentrations of water-insoluble components introduced into the test chamber, either of these factors could have been responsible for the weakened effect. Fractions of the odor obtained by washing with either sodium hydroxide or hydrochloric acid did not influence the results of the tests. This suggests that the essential components of the olfactory signal include both basic and acidic compounds.

Identification of some volatile compounds in the odor of fecal pellets of the rabbit,Oryctolagus cuniculus.

A study has been made of the volatile constituents in the vapors collected from fecal pellets of wild rabbits,Oryctolagus cuniculus. Measurements of changes in the heart rates of adult male rabbits exposed to the effluent of a gas chromatographic capillary column were used to indicate the presence of compounds which may be of behavioral significance. Combined with the use of high-resolution columns in gas chromatography and gas chromatography-mass spectrometry, this approach has implicated several classes of compounds, including volatile fatty acids and phenols, which in certain absolute or relative concentrations may be involved in the formation of signals of territorial importance.

Effect of volatiles collected above fecal pellets on behavior of the rabbit,Oryctolagus cuniculus, tested in an experimental chamber : II. Gas chromatographic fractionation of trapped volatiles.

Gas chromatographic techniques were used to trap and fractionate the volatile substances emanating from the fecal pellets of wild rabbits,Oryctolagus cuniculus, which incorporate the odor of the anal gland secretion. The volatiles were bubbled through water, trapped on Chromosorb 105, transferred to Silicone SF96 traps, passed through a GC capillary column, and retrapped on SF96. The behavioral effect of trapped and fractionated volatiles was determined in four series of bioassays involving 51 adult, male, wild-type rabbits in 112, ten-minute tests. The bioassay was based on the demonstration of territorial confidence by the rabbits under the influence of their own odor. In the bioassays of the unfractionated volatiles eluted from Chromosorb 105 and SF96 traps the animals were significantly more confident in the presence of the volatiles from their own fecal pellets. One of the two fractions of the total volatiles was ineffective while the effect of the other was less than that of the unfractionated odor. The results demonstrate that the gas chromatographic techniques can be used to manipulate complex mammalian odors with precision. Combined with a discriminative bioassay, this opens up the possibility of identifying the specific combinations of volatile substances involved in the formation of olfactory signals.