Methods of inducing and assessing positive affective states in juvenile male Wistar rats

The importance of animals having positive experiences is essential for animal welfare as the balance of positive and negative affective states can be seen as critical to determining an animal’s quality of life. However, a traditional focus on alleviating negative affective states has meant that the complete assessment of animal welfare is hindered by a relative lack of validated models of positive affective states. This disparity between the measurement of positive and negative affective states is particularly evident in rodents used for research. With over 1.9 million rats used for research purposes in the UK, USA and EU per annum as of 2018, contribution towards this gap in knowledge on positive affective states is vital to improving their welfare. As such, the aims of this doctoral research were twofold; first, to develop models of inducing a positive affective state in rats, and second, to investigate whether play is sensitive to the manipulation of affective state as an indicator of PAW. Playful handling is a recently developed approach to positive interactions between humans and rats which aims to incorporate the diversity and unpredictability of juvenile rat social play. To validate the use of playful handling as a method of inducing a positive affective state in juvenile male rats, two independent cohorts were either playfully handled or control handled over successive days, as individuals or pairs. In response to playful handling, rats showed increased production of 50-kHz ultrasonic vocalisations (USVs) compared to control rats, a type of vocalisation associated with positive affect. Approach behaviour towards the experimenter was higher when rats were playfully handled as individuals compared to controls. I also measured rat preference for a high concentration (20%) of sucrose over water as a prospective behavioural test of positive affect. This was an adaption of the sucrose preference test which is a validated test of negative affect which measures preference for a low concentration (sucrose over water. Rats which were playfully handled as individuals drank more sucrose solution than control rats. Thus, there is potential for using a high concentration sucrose preference test as a measure of behavioural and neural changes induced by a positive experience. This increase in indicators of positive affect provides further evidence for the use of playful handling to induce a positive affective state in rats. Presentation of positively valenced rat-produced USVs in playback paradigms is a novel method of altering affective state. This method is based on the premise that as vocalisations have a communicative function associated with affective state, the presentation of positive vocalisations will stimulate positive emotions in the recipient. Four independent cohorts were used to develop a novel paradigm in which acoustic stimuli could be presented in low-stress conditions. Across these experiments, four acoustic stimuli were used; positively valenced 50-kHz USVs, negatively valenced 22-kHz USVs, White Noise, and Background Noise. Across all four experiments using the novel playback paradigm, the presentation of 50-kHz stimuli increased subject-produced positively valenced 50-kHz USVs compared to Background Noise. A single presentation of White Noise also increased 50-kHz USVs to match those produced when presented with 50-kHz compared to Background Noise. This effect of White Noise on USV production was not seen after subsequent presentations, with USV production lowering to that of Background Noise on day five. When the speaker was placed on the side of the cage, there was also an increase in approach behaviour in response to 50-kHz stimuli compared to 22-kHz and Background Noise. The differences in measures of affective state in response to the auditory stimuli across cohorts provide evidence that playback is a promising method of stimulating a positive affective state in rats. Play behaviour is proposed to be a promising indicator of positive emotions and welfare in domesticated animals. To investigate whether play changes with the manipulation of affective state, home cage behaviour was recorded before and after experiencing successive days of handling treatments or presentation of acoustic stimuli. After five days, there was an increase in play in the home cage prior to the experience in response to both playful handling and the playback of 50-kHz USVs. As these effects were specific to behaviour in the home cage occurring before the positive experience, this suggests that play may in part reflect a form of anticipatory behaviour. In the playback paradigm, the absence of a human-rat interaction also allowed the assessment of play during the presentation of acoustic stimuli. After five days of presentation with 50-kHz USVs, rats expressed more social play than those exposed to control stimuli. These results suggest there is a positive relationship between concurrent and pre-existing positive affective state and play. Overall, this work provides support for the use of playful handling and presentation of 50-kHz USVs in a novel playback paradigm as methods to induce a positive affective state in rats. The successful manipulation of indicators of affective state allowed the investigation of play as an indicator of positive affect. As play was sensitive to increases in positive affect, there is evidence that play represents more than just the absence of a negative affective state and supports the use of play as an indicator of positive welfare.