Rapid technical advances in the field of computer animation (CA) and virtual reality (VR) have opened new avenues in animal behavior research. Animated stimuli are powerful tools as they offer standardization, repeatability, and complete control over the stimulus presented, thereby “reducing” and “replacing” the animals used, and “refining” the experimental design in line with the 3Rs. However, appropriate use of these technologies raises conceptual and technical questions. In this review, we offer guidelines for common technical and conceptual considerations related to the use of animated stimuli in animal behavior research. Following the steps required to create an animated stimulus, we discuss (I) the creation, (II) the presentation, and (III) the validation of CAs and VRs. Although our review is geared toward computer-graphically designed stimuli, considerations on presentation and validation also apply to video playbacks. CA and VR allow both new behavioral questions to be addressed and existing questions to be addressed in new ways, thus we expect a rich future for these methods in both ultimate and proximate studies of animal behavior.

The ability to recognize and distinguish between visual stimuli is fundamental for everyday survival of many species. While diverse aspects of cognition, including complex visual discrimination tasks were previously successfully assessed in fish, it remains unknown if fish can learn a matching-to-sample concept using geometrical shapes and discriminate between images and their mirror-image counterparts. For this purpose a total of nine Malawi cichlids (Pseudotropheus sp.) were trained in two matching-to-sample (MTS) and three two-choice discrimination tasks using geometrical, two-dimensional visual stimuli. Two out of the three discrimination experiments focused on the ability to discriminate between images and their mirror-images, the last was a general discrimination test. All fish showed quick associative learning but were unable to perform successfully in a simultaneous MTS procedure within a period of 40 sessions. Three out of eight fish learned to distinguish between an image and its mirror-image when reflected vertically; however none of the fish mastered the task when the stimulus was reflected horizontally. These results suggest a better discrimination ability of vertical compared to horizontal mirror-images, an observation that is widespread in literature on mirror-image discrimination in animals. All fish performed well in the general visual discrimination task, thereby supporting previous results obtained for this species.

The use of computer animation in behavioral research is a state-of-the-art method for designing and presenting animated animals to live test animals. The major advantages of computer animations are: (1) the creation of animated animal stimuli with high variability of morphology and even behavior; (2) animated stimuli provide highly standardized, controlled and repeatable testing procedures; and (3) they allow a reduction in the number of live test animals regarding the 3Rs principle. But the use of animated animals should be attended by a thorough validation for each test species to verify that behavior measured with live animals toward virtual animals can also be expected with natural stimuli. Here we present results on the validation of a custom-made simulation for animated 3D sailfin mollies Poecilia latipinna and show that responses of live test females were as strong to an animated fish as to a video or a live male fish. Movement of an animated stimulus was important but female response was stronger toward a swimming 3D fish stimulus than to a “swimming” box. Moreover, male test fish were able to discriminate between animated male and female stimuli; hence, rendering the animated 3D fish a useful tool in mate-choice experiments with sailfin mollies.

Animal behavior researchers often face problems regarding standardization and reproducibility of their experiments. This has led to the partial substitution of live animals with artificial virtual stimuli. In addition to standardization and reproducibility, virtual stimuli open new options for researchers since they are easily changeable in morphology and appearance, and their behavior can be defined. In this article, a novel toolchain to conduct behavior experiments with fish is presented by a case study in sailfin mollies Poecilia latipinna. As the toolchain holds many different and novel features, it offers new possibilities for studies in behavioral animal research and promotes the standardization of experiments. The presented method includes options to design, animate, and present virtual stimuli to live fish. The designing tool offers an easy and user-friendly way to define size, coloration, and morphology of stimuli and moreover it is able to configure virtual stimuli randomly without any user influence. Furthermore, the toolchain brings a novel method to animate stimuli in a semiautomatic way with the help of a game controller. These created swimming paths can be applied to different stimuli in real time. A presentation tool combines models and swimming paths regarding formerly defined playlists, and presents the stimuli onto 2 screens. Experiments with live sailfin mollies validated the usage of the created virtual 3D fish models in mate-choice experiments.

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