Anosmia In ZnSO₄-Treated Pigeons: Loss of Olfactory Information During Ontogeny and the Role of Site Familiarity in Homing Experiments

Among the many hypotheses put forward to explain pigeon homing (reviewed by Schmidt-Koenig, 1979, 1985, 1987; Wiltschko and Wiltschko, 1978, 1982, 1987; Waldvogel, 1989; Wallraff, 1988a; Papi, 1990), olfactory navigation remains the most contentious. A major source of dispute is the effectiveness of the techniques applied to achieve olfactory deprivation (Wallraff, 1988b) in tests of homing ability in anosmic pigeons.

Intra-nasal irrigation with zinc sulphate (ZnSO₄) is a new, rapid and simple method for olfactory deprivation in pigeons. Treatment with 18% ZnSO₄ solution results in anosmia that persists for at least 5 days. Effects on the pigeons’ optical and acoustical systems could not be detected when tested by the ‘orienting response’ (Schlund, 1991a, 1992). Manipulations with ZnSO₄ caused weight loss of the bulbus olfactorius and damaged the nervus olfactorius in mice (Margolis et al. 1974; Harding et al. 1978), and this could not be excluded for pigeons.

Application of ZnSO₄ alters or destroys the olfactory mucosa and has been found to be effective in hamsters, mice and sea turtles (reviewed by Alberts, 1974). After some time, the olfactory mucosa regenerates, re-establishing olfactory capacity (Smith, 1951; Margolis et al. 1974; Matulionis, 1975, 1976; Harding et al. 1978; Cancalon, 1982).

The present paper reports on a series of experiments designed to test the homing ability of anosmic pigeons. The birds were made anosmic with zinc sulphate. With the help of the ‘orienting response’, we tested the olfactory capabilities of every individual.

Our first series of experiments was designed to test whether anosmic pigeons are able to compensate for the loss of olfactory information when released from familiar sites. For the second experimental series, we reared pigeons anosmically by repeated application of ZnSO₄. Thus, we prevented them from collecting olfactory information during ontogeny and studied how this affected their orientation at unfamiliar sites. In addition, we compared them with a second group of experimental pigeons which were made anosmic just before the critical releases.

Pigeons (Columba livia), between 2 and 5 years old, were housed at our loft near Tübingen (Germany). They had all completed a standard training programme of flights of up to 40km from the cardinal compass directions. The birds had performed numerous single homing flights from various distances and directions in previous years.

In order to familiarise the pigeons with the release sites, they were released as a flock from that site a few days before the critical experiment. We used four sites between 9 and 24km (distance I) and four sites between 54 and 70km (distance II) from the loft, located as symmetrically as possible around it.

Pigeons that had homed from the flock releases were subdivided into three groups as uniformly as possible in terms of age, experience and number. In the critical experiment each individual was released only once.

Pigeons were treated on two successive days with an 18% ZnSO₄ solution: 11.2g of ZnSO₄·H₂O (179.45 g mol⁻¹), 0.1g of Tagat 02 (emulsifier; trade name) and 25g of Frigen 12 in 63.7g of distilled water. Approximately 0.2–0.3ml was sprayed through the choanes into the nasal cavities with an aerosol can.

The day before the critical release and immediately after they had arrived at the loft, the olfactory perception in ZnSO₄-pigeons was tested using cardiac responses. We tested the olfactory capabilities of every pigeon in response to odorous stimuli. Only pigeons individually confirmed to be unable to smell (subsequently called ‘anosmic’) were used for the release and subsequent statistical analysis. Intra-nasal irrigation with 18% ZnSO₄ solution results in anosmia that was found to persist for at least 5 days (Schlund, 1992). For further descriptions of the treatment, the experimental apparatus and the techniques for the olfactory test, see Schlund (1990, 1992).

Control birds were handled like the ZnSO₄-pigeons. Instead of treatment with ZnSO₄ solution, they were sham-treated with a corresponding solution without ZnSO₄.

These were completely untreated pigeons.

We received pigeons at the age of 3–4 weeks from local breeders. On the day of arrival at our loft near Tübingen (Germany), the pigeons were divided into five groups.

Pigeons were reared anosmically by treating them with ZnSO₄ solution on the day of arrival and subsequently once a week. To ensure that the birds were anosmic, the olfactory capabilities of each pigeon were tested every week. Additionally, the olfactory perception in P-ZnSO₄-pigeons was tested immediately upon arrival at the loft after the critical releases.

Controls were treated similarly, except that the control spray, which was delivered once a week, did not contain ZnSO₄. The olfactory capability of each pigeon was tested several times during the experimental period.

Pigeons were made anosmic a few days before the critical release by treating them with ZnSO₄ on two successive days. The second day of treatment corresponded with the time of ZnSO₄-application to the P-ZnSO₄-pigeons. On the following day and immediately after homing, the olfactory capability of every pigeon was tested.

Controls were handled like the short-term ZnSO₄-pigeons, but sham-treated with control spray lacking ZnSO₄.

These were completely untreated pigeons.

As soon as they were able to fly (at the age of 5–6 weeks), pigeons were usually allowed to fly around the loft. When they were about 10 weeks old, we began the standard training programme consisting of eight flock releases from the cardinal compass directions at distances of up to 6km from the loft. The critical releases began at the age of 15 weeks. In the critical experiment, each individual was released only once.

For the critical releases, we used four sites between 15 and 24km from the loft.

The pigeons were released singly, alternating between groups. They were followed visually using 7×50 binoculars with an incorporated magnetic compass until they vanished from sight. The vanishing bearings were recorded to the nearest 1°. Homing times were also recorded. All releases were performed under sunny conditions in 1990 and 1991.

For circular statistics, we considered only vanishing bearings. For each sample and release distance the length (a) and direction (α) of the mean vector were calculated with respect to geographical north. Each sample was tested for uniformity preferences using the Rayleigh test (Batschelet, 1981). For samples with significant directional preferences, we calculated the 95% confidence interval of the mean direction (‘Bootstrap’ with 500 replica; Cabrera et al. 1991). If the 95% confidence interval of the mean direction did not include the home direction, the two directions were considered to differ.

Vanishing bearings of different samples were compared by means of first-order (Watson U²-test) and second-order statistics (Hotelling test: d.f. 2,5).

Homing perfomances were compared using the Mann–Whitney U-test (Siegel, 1956). For this, homing speed per release was standardised by the median of super controls. Pigeons that homed within 3 days after release (lofts were checked every morning and evening) were assigned relative homing speeds: homed on the evening of the releasing day, 0.9; homed the next day, morning, 0.8; homed the next day, evening, 0.7; homed the second day, morning, 0.6; homed the second day, evening, 0.5; homed the third day, morning, 0.4; homed the third day, evening, 0.3; did not home, 0.1.

For each release, standardisation was performed separately before summarising the data per distance and per group. Tests were run on a PC with the help of SAS (1987).

According to the results of heart rate tests after release, two ZnSO₄-pigeons used for releases from familiar sites (distance I and II) and nine ZnSO₄-pigeons in the second experimental series (eight ST-ZnSO₄, one P-ZnSO₄) were able to smell. They were eliminated from the analyses.

The data for all releases are given in Table 1. If pooled with respect to home, ZnSO₄-pigeons, controls and super controls showed directional preference (Rayleigh test). The 95% confidence interval of the direction of the mean vector of all three groups included the home direction (Fig. 1).

The two groups of controls were indistinguishable in comparisons using both first-and second-order statistics. Also, the ZnSO₄-pigeons did not differ from either of the two groups of controls (Table 2).

The homing speed of ZnSO₄-pigeons was slower than the speed of either group of controls. This was true for the single releases (Table 1) as well as for the combined releases pooled with respect to the median of super controls, as shown in Fig. 2 (Mann– Whitney U-test: SC-ZnSO₄: z=4.88, P<0.001; C-ZnSO₄: z=4.16, P<0.001). There was no difference between the two control groups (Mann–Whitney U-test: z=0.67, P>0.05).

Table 3 summarises the principal data for single releases. If pooled with respect to home, it was only the super controls that showed a directional preference (Rayleigh test) and for whom the 95% confidence interval of the direction of the mean vector included the home direction. The distribution of the pooled vanishing bearings of the ZnSO₄-pigeons and C-pigeons, however, did not differ from random (Fig. 3).

The two groups of controls were indistinguishable according to first-order and second-order statistics. Also, there was no difference between the ZnSO₄-pigeons and controls and between ZnSO₄-pigeons and super controls, even though the Watson-U² test approached significance (P<0.1) (Table 2).

As for releases from distance I, the homing speed of ZnSO₄-pigeons was slower than the homing speed of either group of controls (Fig. 4; Mann–Whitney U-test: z=3.64, P<0.001). There was no difference between the two control groups (Mann–Whitney U-test: z=0.77, P>0.05).

For both distances, the vanishing bearings were pooled with respect to the mean of the super controls. No additional information could be gained.

The first time that pigeons were allowed to fly around the loft, 20 of 54 P-ZnSO₄-pigeons were lost (summarised for both years). After the entire training phase, only 32 P-ZnSO₄-pigeons remained for the critical releases.

The principal data for the single releases are summarised in Table 4. Fig. 5 shows the mean vectors for anosmic birds (P-ZnSO4, ST-ZnSO₄) and controls (PC, STC, SC). All three control groups and the P-ZnSO₄-pigeons showed significant directional preferences (Rayleigh test) when results were pooled with respect to home. The 95% confidence interval of the direction of the mean vector of all four groups included the home direction. The distribution of the pooled vanishing bearings of ST-ZnSO₄-pigeons did not differ from random. Table 5 compares the vanishing bearings of the different release groups by means of first-order (Watson-U² test) and second-order statistics (Hotelling test). The orientation of the permanently anosmic pigeons did not differ from that of SC-and PC-pigeons using both first-and second-order statistics. Although there was no difference between the ST-ZnSO₄-pigeons and the controls (Table 5), the results show a tendency towards significance. In addition, there were no differences either between P-ZnSO₄-pigeons and ST-ZnSO₄-pigeons or between any groups of controls.

The relative homing speeds are illustrated in Fig. 6, giving the combined releases pooled with respect to the median of super controls. Table 6 gives comparisons of the relative homing speeds between all groups using the Mann–Whitney U-test. ST-ZnSO₄-pigeons and P-ZnSO₄-pigeons homed more slowly than the controls. Although the homing speed of P-ZnSO₄-pigeons was lower than that of the ST-ZnSO₄-pigeons, there was no difference between the groups. Additionally, there was no difference between super controls (SC) and short-term controls (STC) but, compared with the permanent controls (PC), the homing speed of the STC birds was significantly reduced.

The anosmic ZnSO₄-pigeons released at familiar sites were capable of correct initial orientation only at shorter distances (distance I). In releases from distance II, the distribution of vanishing bearings of anosmic ZnSO₄-pigeons was more scattered than the distribution of vanishing bearings of controls. Homing ability, however, was drastically reduced in ZnSO₄-pigeons of both series. Compared with anosmic ZnSO₄-pigeons released from unfamiliar sites (Schlund, 1992), relative homing speeds of ZnSO₄-pigeons from familiar sites at distances I and II were significantly faster (Mann–Whitney U-test; distance I: z=3.24, P<0.02; distance II: z=3.34, P<0.001). There was no significant difference between the relative homing speeds of control groups.

There is evidence, however, that familiarity with the release site can affect the initial orientation and the homing success of pigeons (Benvenuti et al. 1973; Hartwick et al. 1977; Kiepenheuer, 1982; Lednor and Walcott, 1988; Papi, 1986; Wallraff and Neumann, 1989; Grüter and Wiltschko, 1990; Braithwaite and Guilford, 1991). On the basis of their findings, Wallraff and Neumann fell back on the hypothesis that pigeons rely on two independent homing methods, olfactory navigation and non-olfactory pilotage. Griffin (1952, 1955) and Matthews (1955) considered orientation along familiar visual landmarks as the simplest homing strategy. According to Wallraff and Neumann, the use of visual landmarks should enable homing of anosmic pigeons over distances of 50km and more.

Our findings, however, argue against this hypothesis. In releases from distance II, familiar landmarks did not determine the course of departure of anosmic pigeons and, in releases from both distances, they showed poor homing performances. Benvenuti and Fiaschi (1983) demonstrated that familiarity with the release site does not depend on visual input. Pigeons were homeward-oriented even when made anosmic and wearing frosted lenses. There is no evidence of pilotage using landmarks. At shorter distances, orientation could also be a navigational process.

Thus, the nature of the features which make a site ‘familiar’ remains unknown. This leads back to the idea of the navigational ‘map’ described by Wallraff (1974) and Wiltschko and Wiltschko (1982, 1987), which is hypothesised to be extrapolated beyond the area of immediate experience. By obtaining additional local experience, the pigeons can update their ‘map’. Improving initial orientation and homing appears to depend on this modification of the birds’ map as a result of previous familiarity with the area.

Our findings, however, indicate that familiarity with the release site has no effect on initial orientation in releases from distance II. This may be because the birds do not know the area well enough after only one previous flight. At the release sites located only 9–24km away (distance I), it seems possible that pigeons recognised a site known from former experience, for example during their training flights. So these birds were, in fact, more familiar with the general region around and beyond the test site than were birds released from distance II.

The data obtained with permanently anosmic pigeons from releases in summer 1990 (Fro, Hec) and 1991 (Ber, Ler) are consistent in both years (see Table 4): the vanishing bearings of permanently anosmic pigeons and of all control groups showed a directional preference. The distribution of the pooled vanishing bearings of temporarily anosmic pigeons did not differ from random but, nevertheless, there was no significant difference in the mean vanishing directions between all groups of experimentals and controls (Table 4). This might be an effect of the small sample size of these pigeons because only the data for birds found to be still anosmic upon return to the loft were analysed statistically.

Papi et al. (1972, 1973) advanced a hypothesis of navigation based on an olfactory ‘mosaic’ map. According to this hypothesis, pigeons associate specific odours with the direction of the winds carrying these odours to the loft. Permanently anosmic birds, having been prevented from establishing such an olfactory ‘map’, should not be able to determine the home direction. Nevertheless, our pigeons were capable of doing so, in contrast to pigeons made anosmic by severing the olfactory nerve (Papi et al. 1989). It should be borne in mind that nerve section results in side effects (Wenzel and Rausch, 1977; Wenzel, 1982, 1983). In addition, if pooled with respect to the mean of super controls, the vanishing bearings of P-ZnSO₄-pigeons showed a bimodal distribution (Fig. 7) that corresponds to an orientation along an axis. After doubling the angles, the resulting cluster is well oriented (α=6°, a=0.62, N=21, P<0.005).

These results support the hypothesis of the multifactorial navigational system (Keeton, 1974), which is at present the most widely accepted. According to this hypothesis, the pigeon can use different cues for navigation. The cluster analyses of Ganzhorn (1990) yielded a similar picture, in which pigeons in various regions were assumed to differ in strategies and in the factors they use to orient. In all probability, there is a certain hierarchy in choosing the navigational factors. If a type of olfactory information is missing during ontogeny, a ‘map’ will perhaps be established that depends on the available navigational cues but not on olfactory cues (Wiltschko and Wiltschko, 1985, 1987; Wiltschko et al. 1987). According to this theory, it is not surprising that short-term manipulations with ZnSO₄ caused disorientation. Releases of temporarily anosmic pigeons treated with xylocain showed similar results (Papi et al. 1989), although local anaesthetics have been shown to induce systemic effects (Schlund, 1990) and not to be very dependable (Wallraff, 1988b; Schlund, 1991 b). The learned system of temporarily anosmic birds during rearing depends on a functioning olfactory system that was not available at the critical release.

Homing speed and homing success were drastically reduced in permanently and temporarily anosmic pigeons. The decrease in homing performance of the permanently anosmic pigeons is in contrast to their good initial orientation. This phenomenon is also described by Keeton et al. (1977). Birds obviously ‘knew’ the home direction but were not able to complete the route home. In the P-ZnSO₄ group some of the birds were hunted down by goshawks and many more landed, perhaps because of a reduced motivation for homing rather than because of orientation deficits. Unfortunately, we never received reports about permanently anosmic ZnSO₄-pigeons that did not home. Thus, there is no evidence of the distances they had covered. This discrepancy supports the idea that the navigational strategies are based on two different mechanisms: one for initial orientation at the release site and another for the rest of the homing process.

Furthermore, little is known about the possible side effects of treatment of pigeons with ZnSO₄. In mice, treatment with ZnSO₄ caused weight loss of the bulbus olfactorius (Harding et al. 1978). The action of ZnSO₄ in the olfactory system and even adjacent parts of the brain of pigeons could have effects on learning, motivation, circadian rhythms or magnetic perception (discussed in detail by Schlund, 1992).

It should be emphasised that the results, although suggestive, do not prove olfactory navigation. The experiments do not provide evidence that odorous substances are involved in homing or that the experimental interference interacts exclusively with olfactory functions.

We wish to thank Professor Dr K. Schmidt-Koenig and Dr J. U. Ganzhorn for reading this manuscript. Dr J. Burkhardt and F. Scharfe helped greatly in preparing the manuscript. M. Franck and H. Kaupp developed the appropriate zinc sulphate solutions in aerosol cans. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 307).