In the present work, the influence of natural zeolites on the parameters of defensive behaviour was analysed in experiments with white laboratory rats and a modular device. The obtained data suggested that among the experimental individuals under conditions of tool stress, 76% preferred the food that contained a mineral additive in the form of a crushed zeolite, while the indicators of behavioural activity were optimized in the given category of animals.
Rats ; Defensive behaviour ; Zeolite
There are many contemporary hypotheses concerning the cause and effect geophagy instinct in mammals (Panichev, 1990 ). One of the theories that seeks to explain this phenomenon is based on the assumption that the geophagy phenomenon with its diverse manifestations is an instinctual drive of the organism to achieve the vital constants of material composition and functions of the various physiological systems, which may diverge due to various adverse environmental stressors, with natural minerals (Panichev and Golokhvast, 2009 ).
Several studies reported that animals consume natural minerals in stressful situations involving psychophysiological disadaptation (Soloviev et al ., 2004 ; Panichev, 1990 ; Panichev and Golokhvast, 2009 ; Golokhvast et al ., 2014 ).
The purpose of this work was to study the behavioural parameters of laboratory animals (average of typological group) that prefer feed with the addition of crushed zeolites of Sakhalin origin.
Experiments (second phase) were conducted for 48 h on white outbred male rats weighing 180–230 g that were kept under standard vivarium conditions.
The first experiment focused to the selection of animals according to the typological characteristics through a previously established method (Golokhvast et al., 2014 ).
Content and all procedures with experimental animals were carried out in compliance with the requirements of the WSPA. The experiment involved only those animals that preferred the feed with added ground zeolite (of Sakhalin origin). In total, such animals comprised 76% of the overall number of the observed rats. All of the tested individuals belonged to the II (medium) behavioural type according to our classification (Grigor'ev et al., 2007 ).
According to the previously established methods (Golokhvast et al., 2010 ), zeolites were milled using the ultrasonic homogenizer Bandelin Sonopuls 3400 (product of Bandelin, Italy) for 10 min, and the particle size of the zeolite was approx. 10 μm. The studied zeolite was added to the feed to comprise 5% of the weight of the dry feed.
To analyse the animals' abilities to attain the food reinforcement, a developed modular unit was employed (Batalova et al., 2009 ). An aversive water obstacle was placed in its entrance tunnel (Fig. 1 ).
Scheme of the modular device used for the defensive behaviour study of the laboratory rats.
The floor of the starting compartment was covered by a trellis that was supplied with an impulse threshold current of 0.1–0.2 Ma. In the first phase of the survey, the experimental animals exhibited an instrumental active avoidance reflex (IAAR), and all of the exits in this case were opened. After the IAAR of all laboratory rats was formed, the second stage of observation started. In this stage, in order to exit, the rats had to open the cell door by pushing the pedal or walking through the water, with further recording of motivation energy (seek time (ST), search intensity (SI), and cognitive characteristics (cognitive indicator (CI)). ST reflected the total time that was spent in finding all four compartments. SI described in quantitative terms the frequency of runs carried out for a certain period of time, and CI referred to the ratio of error-free runs to the erroneous ones.
The feed preference was given to animals after the first phase of testing on a background of afferentation formed by reactive situational stress.
Statistical processing of the research results was carried out by methods of variation statistics that assessed the statistical values and differences of samples examined by Students t-test using the Biostat software (version 5.1) (Glants, 1999 ). Differences in the two groups were considered plausible at a significance level of 95% (p < 0.05).
Indicators of the motivation energy (ST and SI) displayed a gradual decrease during the three days of testing (Table 1 ).
|Indicators||Group||First day||Second day||Third day|
|ST (s)||Control||234.7 ± 48.3||215.8 ± 25.0||193.3 ± 14.8|
|Zeolite||215.4 ± 29.5||187.8 ± 31.3||115.7 ± 14.7+ *|
|SI (Units)||Control||37.4 ± 2.1||28.4 ± 1.7||25.7 ± 3.1|
|Zeolite||34.8 ± 5.7||30.2 ± 2.4||21.9 ± 4.3+|
|CI (%)||Control||34.5 ± 18.3||39.9 ± 12.8||47.7 ± 8.4|
|Zeolite||51.8 ± 10.2||67.3 ± 10.0*||73.0 ± 11.3*|
Note: * — values at р < 0.05 compared to control; + — values at р < 0.05 compared to the first testing day.
The ST in the control group declined 1.2 times over 3 days of observation without a significant difference, while in the experimental group, it decreased 1.9 times (p < 0.05). On the first day of the experiment, the difference in the control indicators of ST amounted to 19.3 s and 28.0 s on the second day (p > 0.05). On the last day of testing, a significant difference between the ST values was noted in the group that received zeolite, with similar values to the control sample. In this case, the decline over 77.6 s of the ST was observed (p < 0.05).
By the end of the test, the SI of the control sample decreased 1.5 times compared to the first day (p < 0.05), while the SI of the experimental group decreased 1.6 times (p < 0.05). On the first day of observation, the difference in obtained values of the experimental group compared to the control remained almost unchanged at 2.6 units, during the second day it was 1.8 units, and on the third day, it was 3.8 units (p < 0.05 all days). Despite the lack of a significant difference, in general there was a tendency toward optimization of this parameter, with a leading position in the group of animals that received the mineral supplement.
CI characterizing cognitive behavioural activity in the experimental group increased substantially, although it did not always have a reliably significant difference. Thus, in the first day, it increased by 17.3% (p > 0.05), in the second day by 27.4% (p < 0.05), and in the third day by 25.3% (p < 0.05). Comparing the ST values of the last day with the first testing day, accurate significant differences were not observed in the control group or in the test group (with a decrease of 1.4 in both cases, p > 0.05).
Changing stability of the observed parameters in the control and experimental laboratory rats could be explained by the peculiarities of the behavioural types of these animals. The representatives of the II (middle) type are characterized by a relatively stable parameter constancy of the motivation energy and cognitive spheres of behavioural activity and smooth changes under the influence of any internal and (or) external environmental factors. In the present case, initially high values of CI were observed in the control group (34.5%) and in the experimental group that received zeolite (51.8%).
Analysing the overall picture, it should be noted that the animals on a mineral diet had the most ideal values that characterized defensive behaviour. Complex indirect mechanisms of a zeolites influence on the conditioned reflex activity are impossible to explain clearly today. Exposure to adverse environmental factors (including psycho-emotional factors) is likely to cause problems of adaptation, while the ability to behaviourally resolve the vital constants contributes to the regulation of physiological, information and energy processes in living organisms (Panichev and Golokhvast, 2009 ; Golokhvast et al ., 2014 ). Minerals that have pronounced regulating-stabilizing properties for living systems include smectite minerals of the kaolinite group and some types of zeolites, and their favourable activity for the body has been shown by numerous experiments (Golohvast, 2009 ; Soloviev et al ., 2004 ).
Therefore, the voluntary choice by the laboratory animals (medium behavioural type) to eat a diet that included zeolite additives demonstrates its influence over the energy motivation and cognitive indicators of the behavioural activity under the conditions of the instrumental environment with artificially created problematic situations. The demand to use natural zeolites is likely to be instinctive and is aimed at preserving the life of the organism in the constantly changing environment.