Experimental composition for experimental animal behavioristics as well as preparation method and application of experimental composition

Abstract

The invention relates to a composition for a behavioral experiment of an experimental animal. The composition comprises the following components in percentage by mass: 50-70% of cane sugar, 20-30% of milk powder and 5-20% of glutinous rice flour. The animal training period is effectively shortened, the conventional longer training time is compressed, the behavior index can more sensitively reflect the experimental intervention effect, and the stable operation mode ensures the data reliability under the condition of prolonging the test time or complex experimental paradigm, so that the experimental efficiency is improved. And a solid foundation is laid for other refined behavioral analysis.

Description

Technical Field

[0001] This invention relates to the field of animal feed, and more particularly to a composition, preparation method and application for experimental animal behavior studies. Background Art

[0002] To this day, operant conditioning, established by American scholar B.F. Skinner in the 1930s based on classical conditioning, remains an important behavioral research method in cognitive studies. The principle of reward-operant conditioning tasks is that the animal first performs a voluntary action (response, R), followed by the presentation of a positive reinforcing stimulus (such as sugar water, a food pellet, etc.) as a consequence (outcome, O), thus reinforcing the action. This is accompanied by the presentation of a neutral stimulus (stimulus, S), which transforms into a conditioned stimulus with a predictable behavioral consequence. The animal performs an action in response to the anticipated conditioned stimulus to obtain the reward, forming a complete Skinner operant conditioning (S-R-O). Compared to classical Pavlovian conditioning experiments, operant conditioning experiments incorporating an action task are more complex and varied, representing a co-learning method where the animal learns the consequences of its own actions. In operant conditioning, animals actively adapt to and manipulate their environment to satisfy their needs.

[0003] In operant conditioning research, the use of rodents (such as rats and mice) as experimental subjects is a well-established paradigm. Because rodents share similar physiological structures with humans, and their nervous system structures (such as the cerebral cortex and reward pathways) are highly homologous, their neural response patterns to reward stimuli can be compared to human cognitive mechanisms. Furthermore, rodents face fewer ethical controversies compared to primates, their genomes are easier to edit, and this facilitates the integration of molecular biology techniques to explore the relationship between genes and behavior.

[0004] In operant conditioning experiments on rodents, the positive reinforcement stimuli, or reward substances, are mainly divided into solid and liquid reward substances. Solid rewards are often tablet-shaped or granular, delivered by a stepper motor-driven wheel, with one or several tablets given. Commonly used are pellet feed or sugar pills. Liquid reward substances are provided by a peristaltic pump, which can dispense them once or multiple times. Commonly used are diluted sucrose water, milkshakes, or condensed milk. Some experiments use light and sound stimuli as auxiliary signals to indicate whether the animal's operation is correct or incorrect. Signal lights in the food dispensing area can indicate the distribution of rewards or trigger the start of the experiment.

[0005] In operant conditioning experiments, the selection of reward substances is a crucial step in experimental design, as their properties and quality directly affect the intensity of animal motivation, behavioral stability, and the reliability of experimental data. Conventional reward substances, such as ordinary feed pellets or sucrose solutions, while inexpensive and readily available, are prone to sensory adaptation and reward fatigue in animals after prolonged use as behavioral rewards due to their monotonous taste and lack of sensory stimulation. This diminishing reward valence leads to a gradual decrease in the intensity of motivation for the animal's operant behavior, manifesting as prolonged response latency, decreased accuracy, and increased behavioral variability. Particularly in experimental paradigms requiring continuous multi-day training or single long-duration tests, this limitation of traditional reward substances significantly shortens the effective data collection window, reducing experimental efficiency and potentially introducing additional data variability due to motivational fluctuations, thus affecting the internal validity of the experimental results. Therefore, developing composite reward substances with dynamic sensory characteristics, nutritional balance, and the ability to maintain long-term motivational stability has become an important research direction for improving the quality of operant conditioning experiments. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a composition, preparation method, and application for experimental animal behavior studies.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a composition for experimental animal behavior studies, the composition comprising the following components by weight percentage: 50-70% sucrose, 20-30% milk powder, and 5-20% glutinous rice flour.

[0009] In some embodiments, the experimental animal is a rodent.

[0010] In some embodiments, the composition comprises the following ingredients by weight percentage: 65% sucrose, 20% milk powder, and 15% glutinous rice flour.

[0011] In some embodiments, the composition comprises the following ingredients by weight percentage: 60% sucrose, 30% milk powder, and 10% glutinous rice flour.

[0012] In some embodiments, the composition comprises the following ingredients by weight percentage: 55% sucrose, 25% milk powder, and 20% glutinous rice flour.

[0013] The second aspect is a reward substance for experimental animal behavior studies, wherein the active ingredient of the reward substance is the composition for experimental animal behavior studies described in the first aspect.

[0014] In some embodiments, the reward substance may further include edible flavorings. Through the synergistic effect of the flavorings and the composition itself, the palatability of the reward substance is further enhanced, especially for experimental animals under stress or with low appetite. This effectively increases their desire to actively obtain the reward, thereby improving animal cooperation and task completion quality in behavioral experiments.

[0015] In some embodiments, the reward substance may also include food coloring. It is important to note that the selected food coloring must undergo safety verification to ensure that it does not have any additional impact on the physiological state or behavioral performance of the experimental animals, thereby guaranteeing the reliability of the experimental results.

[0016] In some embodiments, the method for preparing the reward substance is as follows:

[0017] The raw materials are combined in proportion and then finely ground to ensure the particle size and uniformity of the raw materials.

[0018] The pre-treated raw materials, excluding glutinous rice flour, are thoroughly mixed at a temperature below 30°C to form a uniform mixture 1.

[0019] Add 1 / 3 to 1 / 2 of the glutinous rice flour to distilled water, stir well, heat to 70-90℃ and stir until the solution becomes transparent, viscous and without obvious particles. Continue stirring and add mixture 1 to obtain the final mixture.

[0020] The mixed feed is pelleted, dried, and cooled, and then screened to obtain pelleted feed that meets the specifications.

[0021] In some embodiments, the experimental animal is a rodent.

[0022] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0023] This invention, through its unique and scientifically formulated ingredient ratios, offers the following advantages: First, it shortens the animal training cycle, compressing the conventionally longer training time; second, it improves the signal-to-noise ratio of experimental data, enabling behavioral indicators to more sensitively reflect the effects of experimental interventions; most importantly, this stable operating mode ensures data reliability under extended testing times or complex experimental paradigms, laying a solid foundation for further refined behavioral analyses (such as extinction experiments, progressive ratio tests, etc.). Attached Figure Description

[0024] Figure 1 This refers to the appearance of the reward substance finally produced according to the formulation of Example 1 of the present invention. DETAILED DESCRIPTION

[0025] To make the objectives, technical solutions, and advantages of this application clearer, the application is described and illustrated below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. All other embodiments obtained by those skilled in the art based on the embodiments provided in this application without inventive effort are within the scope of protection of this application.

[0026] Obviously, the accompanying drawings described below are merely some examples or embodiments of this application. Those skilled in the art can apply this application to other similar scenarios based on these drawings without any creative effort. Furthermore, it is understood that although the efforts made in this development process may be complex and lengthy, for those skilled in the art related to the content disclosed in this application, any changes to design, manufacturing, or production based on the technical content disclosed in this application are merely conventional technical means and should not be construed as insufficient disclosure of the content of this application.

[0027] The types and sources of the raw materials used in the embodiments of the present invention are shown in Table 1 below. Unless otherwise specified, the other substances are all commercially available conventional products.

[0028] Table 1

[0029]

[0030]

[0031] Pellet feed preparation

[0032] The formulations and ingredients used in Examples 1-3 and Comparative Examples 1-5 of this invention are shown in Table 2. The feed pellets were then prepared according to the following method:

[0033] Step 1: Raw material pretreatment

[0034] According to the formula proportions in Table 2, the raw materials are sieved to remove impurities and large particles, ensuring the particle size and uniformity of the raw materials. Through fine grinding, the glutinous rice flour should pass through a 100-mesh sieve with a pass rate of over 98%, the milk powder should pass through an 80-mesh sieve with a pass rate of over 98%, and the sucrose should pass through a 60-mesh sieve with a pass rate of over 99%.

[0035] Step 2: Mixing

[0036] Add the pre-treated raw materials (except glutinous rice flour) sequentially to a mixer while maintaining a temperature below 30°C. Set the mixer speed to 80 rpm and the mixing time to 5 minutes to ensure thorough and uniform mixing, forming a homogeneous mixture 1. Then, place the weighed glutinous rice flour in a container and add an appropriate amount of distilled or deionized water (1 / 3-1 / 2 the volume of the glutinous rice flour). Stir well and place the container on a heating device, heating to 70-90°C while continuously stirring until the solution becomes transparent, viscous, and free of noticeable granules. Continue stirring and add mixture 1 to obtain the final mixture.

[0037] Step 3: Granulation

[0038] The mixed raw materials are put into the pellet mill. The screw speed of the pellet mill is adjusted to 50 rpm, and the die aperture is selected as 3 mm according to the particle size.

[0039] Step 4: Drying and Cooling

[0040] After pelleting, the feed pellets are spread evenly on a tray and placed in a drying oven. The drying temperature is set at 45℃ and the drying time is 4 hours to reduce the moisture content of the pellet feed to below 10%. After removal, the pellet feed is placed in a ventilated room to cool to room temperature to improve the stability and storability of the pellet feed.

[0041] Step 5: Screening and Packaging

[0042] The cooled pelleted feed is screened by a vibrating screen to remove unqualified broken pieces and oversized particles, and the pelleted feed that meets the specifications is selected as reward material for subsequent experiments.

[0043] Table 2

[0044]

[0045]

[0046] Performance testing methods

[0047] The performance evaluation of Examples 1-3 and Comparative Examples 1-5 in this invention was completed by Skinner box operation reward experiment.

[0048] Forty-eight 10-week-old C57BL / 6J wild-type mice were selected as experimental animals. The females weighed 18g±2g and the males weighed 22g±2g, with half being females and half being males.

[0049] The experiment is divided into the following stages, and the specific experimental steps are as follows:

[0050] 1) Adaptation feeding period: For the first 7 days of training, mice were housed in individual cages under the following conditions: temperature 20-22℃, relative humidity 40%-70%, and a circadian rhythm of 12 hours of light and 12 hours of darkness. Mice were fed irradiated sterilized maintenance feed (from Jiangsu Xiehe Pharmaceutical Biotechnology Co., Ltd.). Daily food intake and body weight were recorded, and the animals' mental state was observed.

[0051] 2) Training Phase 1: 48 mice were divided into 8 groups, with 3 females and 3 males in each group. Each group was placed in a Skinner box and allowed to acclimatize in darkness for 5 minutes. Then, the box lights were turned on, and every 30 seconds, a solid reward substance (3mm x 3mm, cylindrical) was introduced. Figure 1 (As shown) The solid reward particles were delivered to the feeding trough to train the mice to learn the location of the solid reward particles. Each training session lasted 30 minutes, and the mice were trained once a day for 3 consecutive days.

[0052] 3) Training Phase 2: Starting on the third day, mice were trained to press a lever to receive a food reward. The mice were placed in a Skinner box and allowed to acclimatize to the darkness for 5 minutes. Then, the room light was turned on and turned off after 5 seconds. The indicator light above the lever was illuminated, and the lever was popped out, awaiting the mouse's press. If the mouse did not press the lever, it was waited for to press it. When the mouse touched and pressed the lever, the room light was turned on, the lever was retracted, and a quantitative reward granule was delivered to the food trough. After 25 seconds, the room light was turned off for 2 seconds, and the next cycle began. Each training session lasted 30 minutes, once a day for a total of 7 days. On the 8th day, the number of times the mouse pressed the lever within 30 minutes was recorded.

[0053] The results are analyzed as follows:

[0054] Table 1 shows the body weight and food intake of mice during the acclimatization period. The experimental data in Table 1 show that the average daily food intake of the experimental group mice fluctuated by no more than 10% during the 7-day acclimatization period, exhibiting a stable feeding pattern. Meanwhile, a comparison of weights before and after the acclimatization period revealed that the body weight of all experimental group mice increased.

[0055] These data indicate that the mice used in this invention exhibited minimal stress response to the new environment during the adaptation feeding period, successfully completing the environmental adaptation process. More importantly, the stable food intake and healthy weight gain demonstrate that the mice were in an ideal nutritional state. This effectively eliminates differences in motivation levels caused by fluctuations in hunger, providing a standardized behavioral basis for subsequent Skinner box manipulator reward experiments. This rigorous adaptation feeding control ensures that the behavioral responses of the experimental animals during the testing period primarily reflect their motivational characteristics towards the reward substance, rather than being driven by basal metabolic needs, thereby significantly improving the reliability and reproducibility of the experimental results.

[0056] Table 1

[0057]

[0058]

[0059]

[0060] Table 3 shows the number of times the mice pressed the lever within 30 minutes on day 8. As can be seen from Table 3, compared to Comparative Examples 1-5, when Examples 1-3 were used as the reward substance, the mice reached a relatively high daily lever pressing frequency earlier, thus performing the Skinner box experiment better. This indicates that the mice had a higher acceptance of Examples 1-3, formed a conditioned reflex more quickly, and were more conducive to conducting behavioral experiments on mice.

[0061] Table 3 records the average behavioral data (rounded) of each group of mice pressing the lever during the 30-minute test period on day 8 of the experiment. Data analysis shows that the experimental groups using Examples 1-3 as reward substances exhibited significant behavioral advantages: they achieved a relatively high operation frequency in the early stages of the test (the first 3 days) and maintained a stable response rate throughout the entire test period. Finally, on day 8, the total number of presses in 30 minutes reached 40-47, significantly higher than that of the control groups 1-5. This phenomenon indicates that the mice in the Examples 1-3 groups were able to establish the behavioral association of "lever pressing - reward acquisition" more quickly, and their conditioned reflexes formed earlier than those in the traditional reward substance groups.

[0062]

[0063] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for experimental animal behavior studies, characterized in that, The composition contains the following ingredients in weight percentage: 50-70% sucrose, 20-30% milk powder, and 5-20% glutinous rice flour.

2. The composition according to claim 1, characterized in that, The experimental animals were rodents.

3. The composition according to claim 1, characterized in that, The composition contains the following ingredients by weight percentage: 65% sucrose, 20% milk powder, and 15% glutinous rice flour.

4. The composition according to claim 1, characterized in that, The composition contains the following ingredients by weight percentage: 60% sucrose, 30% milk powder, and 10% glutinous rice flour.

5. The composition according to claim 1, characterized in that, The composition contains the following ingredients in percentage by weight: 55% sucrose, 25% milk powder, and 20% glutinous rice flour.

6. A reward substance for use in experimental animal behavior studies, characterized in that, The active ingredient of the reward substance is the composition for experimental animal behavior as described in any one of claims 1-5.

7. The reward substance according to claim 6, characterized in that, The reward substances also include food flavorings.

8. The reward substance according to claim 6, characterized in that, The reward substances also include food coloring.

9. The reward substance according to claim 6, characterized in that, The method for preparing the reward substance is as follows: The raw materials are combined in proportion and then finely ground to ensure the particle size and uniformity of the raw materials. The pre-treated raw materials, excluding glutinous rice flour, are thoroughly mixed at a temperature below 30°C to form a uniform mixture 1. Add 1 / 3 to 1 / 2 of the glutinous rice flour to distilled water, stir well, heat to 70-90℃ and stir until the solution becomes transparent, viscous and without obvious particles. Continue stirring and add mixture 1 to obtain the final mixture. The mixed feed is pelleted, dried, and cooled, and then the pellets that meet the specifications are screened out.

10. The reward substance according to claim 6, characterized in that, Its features are, The experimental animals were rodents.

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