experimental period were analyzed by One-way ANOVA. Experiments were repeated Ž
. with increasingly higher concentration or lower pH of the test compound until a
Ž .
treatment was found that was significantly different p - 0.05
from the control Ž
. water-only group for one or more of the recorded parameters. When a p - 0.05 was
obtained, differences between individual treatment means were determined by least Ž
. significant difference LSD . If a treatment mean was significantly different from the
control mean by LSD, the experimental series was stopped. In the case that there was a Ž
. significant ANOVA p - 0.05 , but the LSD did not indicate that a treatment mean was
significantly different from the control mean, the trial was repeated with the same test solution concentrations or pH. Repeated data sets included sodium chloride on the
non-preferred side at concentrations of 0.12, 0.16, 0.18 mol l
y1
; citric acid buffer on the non-preferred side at pH 5.0; sucrose on the non-preferred side at concentrations of 0.45
and 0.56 mol l
y1
; and sucrose on the preferred side at 0.36 and 0.45 mol l
y1
. Data from Ž
. these replicate experiments were analyzed using a general linear model GLM with
treatment, repetition, and the treatment–repetition interaction as main effects. In most cases, the effects of both the repetition and the interaction were not significant
Ž .
p 0.20 , and the data were pooled and analyzed by One-way ANOVA and LSD Ž
means comparisons. In some cases sucrose on preferred side at concentrations of
y1
. 0.36and 0.45 mol l
for non-preferred and Proportion parameters , repetition accounted Ž
. for sufficient variation p - 0.20 and was left in the model. In these cases, differences
between control and test measures were determined by single degree of freedom contrasts. To compare the effects of different solution concentration within the experi-
mental series, the treatment means were standardized by expressing the consumption parameter means of treatment birds as a percentage of the mean of the same parameter
of the control birds.
Similar threshold levels were found when the test solutions were placed on the preferred and non-preferred sides so we desired to determine which of these two
experimental approaches were most efficacious. Because of variation in the sample size Ž .
of the treatment groups n and in the standard deviations, the data were analyzed by Ž
. power statistics using JMP SAS, Cary, NC in order to compare the relative effective-
ness of placing the flavor solution on the preferred vs. non-preferred sides. The power was calculated using all data from each side for the threshold concentration or pH.
Power calculations resulted in a probability of achieving a p s 0.05 for a range of values for n. These calculations used D, the effect size or the separation of the means,
and S, the standard deviation of the data set, to determine these probabilities. The more
Ž .
powerful experimental design test compound in preferred or non-preferred side was determined by which had the higher probability of being significant for n ranging from
12 to 52 in increments of 4.
3. Results
3.1. Equilibrium periods During the equilibrium periods, initial side- preferences were monitored. Preference
Ž .
Ž ratios ranged from - 0.1 strong preference for side one to 0.9 strong preference
. for side two . The preference ratios appeared to be equally distributed between birds
both before and after exposure to chemical stimuli. The average preference ratio for the whole flock and within treatment groups was typically near 0.5. While it was not tested,
it appeared as if side-preference ratio may relate to the location of the bird in the cage
Ž battery i.e. birds on the left side of the battery appear to prefer the right water bottle and
. vice-versa
3.2. Experiment 1: sodium chloride The sensitivity of cockatiels to a salty substance was first tested by placing a sodium
chloride solution on the non-preferred side and purified water on the preferred side. The eight test solutions of sodium chloride had concentrations ranging from 0.02 to 0.18 mol
y1
Ž .
y1
l Fig. 1a . At 0.12 mol l
, none of the consumption parameters differed signifi- cantly from the same parameters of the control group. At 0.16 and 0.18 mol l
y1
, the Ž
. consumption from the treatment non-preferred side sodium chloride solution was
Ž .
significantly less than the consumption from the control non-preferred side water Ž
. p s 0.004 and 0.016, respectively . Additionally, at both of these levels, the treatment
Ž Proportion was also significantly less than control Proportion
p s 0.003 and 0.021, .
respectively . For all birds offered the 0.16-mol l
y1
sodium chloride solution, the total fluid Ž
. consumption was significantly less than that of the control p s 0.029 . The same trend
of a decreasing total consumption occurred for the 0.18-mol l
y1
sodium chloride group, Ž
. but this result was not significant p s 0.085 .
Based on the results of Experiment 1a, sodium chloride was placed on the preferred
y1
Ž .
side at 0.16 and 0.18 mol l Fig. 1b
and purified water was placed on the
y1
Ž non-preferred side. At 0.18 mol l
, the consumption from the preferred side sodium .
chloride solution was significantly less than the consumption from the control preferred Ž
. Ž .
side water p s 0.011 . Additionally, the consumption from the non-preferred side
Ž .
water was significantly greater than the consumption from the control non-preferred Ž
.
y1
Ž .
side water at 0.18 mol l p s 0.018 . Lastly, the Proportion was significantly greater
y1
Ž .
than the Proportion of the control group at both 0.16 and 0.18 mol l p - 0.001 .
Regardless of which side the flavor was placed, the presence of 0.16 mol l
y1
sodium chloride significantly changed the drinking patterns when compared to the control
patterns. Therefore, the taste threshold for cockatiels is between 0.12 and 0.16 mol l
y1
. Because the number of birds used and the magnitude of the response varied for these
Ž
y1
. two experiments, power statistics were applied to the threshold data 0.16 mol l
to determine which experimental procedure had the highest probability of achieving
Ž significance. The three statistically significant parameters consumption from preferred,
. consumption from non-preferred, and Proportion tested indicated that the experimental
Ž .
design was more powerful higher probability of significance when sodium chloride was placed in the preferred bottles.
3.3. Experiment 2: citric acid To determine the sensitivity of cockatiels to pH, a 0.05-mol l
y1
buffer solution of citric acid and sodium citrate was prepared at pH 5.5 and 5.0 using the proportions
Fig. 1. Comparison of the effects of different concentrations of sodium chloride placed on previously Ž .
Ž . established non-preferred a and preferred b sides. The consumption measures of control birds that were
provided water on both sides were normalized to 100 and are indicated by the horizontal line at this value. Ž
. Open bars indicate consumption by treatment groups SEM expressed as a percentage of the control group.
Stars represent significant changes from the control. The numbers in parentheses indicate the sample size.
Ž .
indicated by Gomori 1955 . The solutions were first offered to the birds on the non-preferred side. No significant differences were found between the control group and
Ž .
the pH 5.5 treatment group for any of the consumption measurements Fig. 2a . At pH
Fig. 2. Comparison of the effects of different pH values of a 0.05-mol l
y1
citric acid buffer placed on Ž .
Ž . previously established non-preferred a and preferred b sides. The consumption measures of control birds
that were provided water on both sides were normalized to 100 and are indicated by the horizontal line at Ž
. this value. Open bars indicate consumption by treatment groups SEM expressed as a percentage of the
control group. Stars represent significant changes from the control. The numbers in parentheses indicate the sample size.
Ž .
Ž .
5.0, consumption from the non-preferred side p s 0.025 and the Proportion p s 0.014 were significantly less than the same parameters of the control group.
Next, the buffered citric acid solutions at pH 5.5 and 5.0 were placed on the preferred Ž
. side Fig. 2b . The consumption from the preferred side of the pH 5.0 treatment group
was significantly less than the consumption from the preferred side of the control group. Additionally, the Proportions for pH 5.5 and 5.0 treatment groups were significantly
Ž .
greater than the control group Proportion p s 0.001 . Therefore, the taste threshold for 0.05 mol l
y1
citric acid buffer is likely to be around pH 5.5.
Fig. 3. Comparison of the effects of different concentrations of sucrose placed on previously established Ž .
Ž . non-preferred a and preferred b sides. The consumption measures of control birds that were provided water
on both sides were normalized to 100 and are indicated by the horizontal line at this value. Open bars Ž
. indicate consumption by treatment groups SEM expressed as a percentage of the control group. Stars
represent significant changes from the control. The numbers in parentheses indicate the sample size.
Power statistics were also calculated for the citric acid buffer data. The probability of achieving significance for preferred consumption, non-preferred consumption, and Pro-
portion was calculated and indicated that the experimental design was more powerful when the test solution was placed on the preferred side than on the non-preferred side.
3.4. Experiment 3: sucrose Sucrose was used to determine the threshold level of sweetness. Sucrose was first
tested on the non-preferred side. Ten concentrations ranging from 0.08 to 0.56 mol l
y1
Ž .
y1
were tested Fig. 3a . At 0.45 and 0.56 mol l , the consumption from the non-preferred
side was significantly less than that of the non-preferred side of the control group Ž
.
y1
p s 0.003 . Additionally, at 0.45 and 0.56 mol l , the Proportion was significantly
Ž .
less than the control Proportion p s 0.001 . When sucrose was placed on the preferred side, only 0.36 and 0.45 mol l
y1
were Ž
.
y1
tested Fig. 3b . The non-preferred consumption from the 0.36- and 0.45-mol l groups
were significantly greater than the non-preferred consumption of the control group Ž
. p s 0.0217 and p - 0.0001, respectively . Also, the non-preferred consumption from
0.36 mol l
y1
was significantly less than the non-preferred consumption from 0.45 mol
y1
Ž .
y1
l p s 0.0190 . Additionally, the Proportion for the 0.36- and 0.45-mol l
groups Ž
. were significantly less than the control Proportion p s 0.0168 and 0.0005, respectively .
The sucrose on the preferred side series was the only experimental series in which a Ž
main effect of repetition was significant. For the two consumption parameters non-pre- .
ferred consumption and Proportion for which repetition was significant, the first
Ž .
repetition was significantly less than the second p s 0.0018 and 0.0047, respectively .
Again, power statistics were used to determine whether the placement of the sucrose taste stimulus on the preferred or non-preferred sides was more powerful. For the
0.36-mol l
y1
treatment data, the three tested consumption parameters were all more readily detected when the solution was placed on the non-preferred side.
Additionally, because of the large number of repetitions using the same birds, the data for sucrose on the non-preferred side was subjected to a test to determine if the
birds were habituating to the increasing sucrose concentrations. A GLM was run testing the data for effects from the repetition or from the treatment by repetition interaction. No
significant effects were found for any of the consumption measures, indicating that the birds were not habituating to the sucrose solution over time.
4. Discussion
