Effect of pair housing on behaviour and gut microbiota of pre-weaned dairy calves

Pair housing is a viable alternative to individual housing for pre-weaned dairy calves, yet aspects like the evolution of behavioural synchronisation and of gut microbiota over time are still poorly understood. This study aimed at evaluating lying synchronisation and gut microbiota composition in pre-weaned dairy calves housed in pairs or individually. 56 Friesian female calves were enrolled from two commercial dairy farms in Northern Italy and housed from birth to 56 days, either individually (n = 28) or in pairs (n = 28; maximum age difference: 24 hours). From two days of age, each calf was fitted with an accelerometer on the right hind leg to monitor lying behaviour. Faecal samples were collected at days 2, 7, 21, 35 and 56 from a subset of individually (n = 12) and pair housed (n = 12) calves to assess gut microbiota composition. Accelerometer data, recorded at 60-second in tervals, were used to estimate lying time as a percentage of the day and as minutes per hour. To identify possible synchronism between pair housed animals and between individually housed calves of similar age, time-series were analysed and overlapped, and the coefficient of determination (R²) was calculated for each day to assess in a preliminary way the correlation degree between time-series of different animals. Daily lying time decreased from 80–82% to 65–67% over the study period, regardless of housing. Pair housed calves exhibited higher R² values, with some pairs showing strong synchrony early on and others increasing from 0.1-0.4 to 0.7-0.8 over time. Individually housed calves showed no such trend (R² not exceeding 0.5). Microbiota analysis showed no significant differences in alpha (within-individual richness and diversity) or beta diversity (between-group rich ness and diversity) between the two housing types, while a farm effect was observed. Significant differences in taxonomic composition were found at 56 days. Individually housed calves exhibited a microbial composition that may reflect a less efficient metabolic state or higher stress (e.g., increased Fusobacterium and Odoribacter), whereas pair housed calves had genera known to be associated with better productive performance and gut health, such as Prevotella and Ruminococcus. Lying synchronization was detectable via accelerometers and might provide an indicator of positive welfare in pair housed dairy calves. Valuable differences were found in taxonomic composition of pair-housed dairy calves’ microbiota: however, studies with larger sample sizes are needed to explore the evolution of taxonomy differences and their correlations with relevant phenotypic traits.