动物实验 z-bio 2021-02-21 558

　　Introduction: The increasing use of probiotics has broadened our knowledge of the effects of intestinal microbial components and bacterial metabolites on neural and behavioral processes through different regulatory pathways. Most research has focused on humans or other mammals. However, in recent years, the use of fish model species has become more and more common, because they are easily kept in artificial captivity, and some of their neural pathways are homologous to those of mammals. Zebrafish is a model species, and the results of its probiotic experiments can be used as a reference for human treatment because they have similar gut microbiota and colonization patterns. The new tank test (NTT) is one of the most widely used experimental procedures designed to test anxiety-related behaviors in zebrafish. It is based on their natural preference that they will swim near the bottom when they are first placed in the new tank. The difference in the time it takes for the fish to leave the bottom of the tank and start exploring other areas is generally thought to reflect the anxiety they experience. A number of studies have used this method to analyze the effects of different substances on fish performance, and a positive correlation with anti-anxiety drugs has been observed. However, the collateral effects of probiotic intake on behavioral responses have not been thoroughly studied. In some cases, the response to stress is reduced, while in other cases there is no direct correlation. Therefore, the mechanism that triggers this response remains unclear. In this study, the effects of the probiotic mixture (1:1) of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 strains on zebrafish exploration behavior have been analyzed, and it has been proved that these two probiotics can improve humans and zebras. The role of fish sperm performance.

　　 This study used two groups of nine adult (8 months old) wild zebrafish (AB strain). Raise all fish in a 3-liter tank and exchange water from a recirculation system equipped with mechanical, chemical and biological filters. Keep the water temperature at 26°C with a 14/10 light-dark cycle. In order to evaluate the effect of probiotics administration on fish behavior, different feeding methods were carried out for each group for 4 months. The control group was fed commercial zebrafish feed twice a day, and the experimental group, in addition to commercial feed, also provided 109 CFU of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 daily. One month before the behavioral test, each fish was anesthetized in 110 mg/L tricaine methanesulfonic acid buffer and marked with visible implanted elastomers.

　　New water tank test: Fill a quadrilateral glass aquarium (20×8×18 cm; length×width×depth) to a total volume of 3.5 L, and use it as an evaluation place. Use a net to place each fish on the bottom of the tank and record its swimming behavior for 6 minutes. After the experiment, the fish was transferred to a recovery bin. The video file was analyzed using NoldusEthovision® tracking software, and a virtual grid was generated to divide the water tank into upper and lower areas. Measure the time spent in each area (expressed as a percentage of the total time), the number of crossings between the areas, the average speed, the total distance moved and the waiting time for the first crossing to the upper area.

　　Result: When a fish is placed in a new environment, its instinct will trigger protective diving at the bottom of the tank, thereby reducing the risk of being attacked by potential predators near the water. The new tank test (NTT) is a common test based on this reaction in zebrafish behavior experiments. Because NTT has a conflict between "safe" diving behavior and "exploratory" swimming behavior, it is possible to study the strength of the fish's diving instinct under specific experimental conditions. Under normal controlled laboratory conditions, usually in the first few minutes of surveillance, fish usually spend more time at the bottom of the tank, exhibiting higher erratic motion and stationary events. After the adaptation period, the animals gradually explore the upper part of the tank. The results showed that four months after adult zebrafish were fed with antioxidants and anti-inflammatory probiotics, their behavior patterns changed drastically compared with the control group. Use Ethovision software to establish two homogeneous virtual partitions to analyze the exploration mode. The results showed that there was a statistically significant difference in preferences for swimming areas (up and down) in the new tank test. When analyzing the heat map, the probiotic-fed animals showed a strong preference for the upper area. On the other hand, standard-fed animals did not show this preference for the upper area. In the control group, only 50% of the test fish showed a preference for the upper area, while 88.89% of the probiotic-fed animals preferred to be near the water. In fact, in the 6-minute new tank test, 7 out of 9 fish in the probiotic group spent 80% of the time in the upper zone. The speed, the total travel distance of each animal, the number of shuttles between areas, and the latency to the first crossing to the upper area were also evaluated. Statistical analysis only records significant differences in average speed. In our experiments, CTRL showed a higher speed compared to fish fed with probiotics. The incubation period for the probiotic group to enter the upper area is very short (no fish lasted more than 10 seconds before exploring the upper area). These data are interesting because they incorporate clear spatial preferences; they indicate clear differences in behavior patterns between groups. Stress can regulate the NTT mode behavior of zebrafish. Some anti-anxiety drugs commonly used to treat human anxiety tend to increase the time zebrafish stay on top of the experimental device. The similarity of the results suggests that the probiotic mixture may have an impact on the anxiety regulation of model animals.

　　 Ingestion of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT73474 can regulate zebrafish behavior after months. (A) Schematic diagram of experimental design. (B) Schematic diagram of the new water tank test (NTT) and the determined analysis area. (C) Ethovision analysis of the trajectories of the two experimental groups (control group and probiotic group). (D) Time spent by the animal in the upper area (%); (E) Average speed (cm/s); (F) Total distance (cm); (G) Number of shuttles (n.) (H) Crossing to the upper area Regional delay.

　　 In short, it is determined that long-term intake (4 months) of Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347 will change the swimming style of the zebrafish model. The results show that feeding adult zebrafish with a mixture of antioxidants and anti-inflammatory probiotics can greatly reduce the zebrafish’s bottoming behavior in the new tank. This main behavioral response is associated with a lower state of anxiety. Therefore, it can be considered that the intake of this probiotic mixture is a beneficial way to treat anxiety.