Environmental enrichment changes rabbits' behavior, serum hormone level and further affects cecal microbiota

Abstract

Many studies have shown that stress is associated with gut microbiota. Environmental enrichment (EE) could reduce stress in farm animals; however, limited information is available on the microbial community composition in rabbits raised with or without EE. This study aimed to identify EE influences on the behavior, serum hormonal levels, and cecal microbiota of rabbits. Two hundred Rex rabbits were segregated randomly within four cohorts (n = 50); reared for 76 d within standardized enclosures (non-enriched) or within cages containing a willow-stick (WS), rubber-duck (RD), or a can of beans (CB). The rabbits' ingestive, rest, locomotion, exploratory, grooming, and abnormal behavior were observed. The serum hormone levels for rabbits were measured, and cecal specimens were sequencedfrom the V3-V4 region using 16S rRNA amplicons. Environmental enrichment increased feeding and drinking time, promoted exploratory behavior, and reduced abnormal behavior in rabbits. Insulin-like growth factor 1(IGF-1) levels of the enriched cohorts were elevated in comparison to the control cohort. Serum cortisol level for CB cohort was markedly reduced in comparison to the control cohort (p < 0.05), while dopamine levels for CB cohort peaked. Further, we found that EE mainly affected the dominant microbiota. Several families, such as Erysipelotrichaceae, Tannerellaceae, Enterobacteriaceae, Burkholderiaceae, and Prevotellaceae were markedly reduced within the CB cohort. Bacteria such as Alloprevotella, Bifidobacterium, Enterobacteriaceae, Parabacteroides, and Erysipelatoclostridium were identified as having negative associations with the presence of serum cortisol. EE influenced rabbit behavior and serum hormonal levels, and CB enrichment was the most suitable for rabbits. Further, cecal microbiota composition and diversity were affected by CB enrichment. These findings suggested that CB could be considered for use in rabbit husbandry.

Keywords: Cecal microbiota; Environmental enrichment; Rabbit behavior; Rabbit husbandry; Rex rabbits.

Figure 1. Environmental enrichment materials.

(A) Willow stick; (B) rubber duck; (C) can of beans.

Figure 2. Effect of different environmental enrichment (EE) materials on rabbits’ behavior (during the observation periods).

(A) Ingestive behavior; (B) Exploratory behavior; (C) Abnormal behavior; (D) Standing, walking, resting, and grooming behavior. CO, control; WS, willow stick; RD, rubber duck; CB, Can of beans. (s) Measured as duration; (times) Counted as instances of behavior. ^∗ p < 0.05, ^∗∗ p < 0.01; values not reported where p > 0.05.

Figure 3. Comparison of cecal microbiota of rabbits in the CO and CB groups.

Mean relative abundance for the 10 best-ranking phyla (A)/top10 families (B) within the CB-and CO -cohort rabbits; (C) Box-plot of cecal microbial β-populations (Wilcoxon rank-sum test, p < 0.01); (D) PCA for rabbit cecal specimens depending upon unweighted UniFrac distances; (E) Similarity-cluster analyses of rabbit cecal specimens through UPGMA. (F) Bacterial taxa differences at the family level; (G) bacterial taxa differences at the genus level. Bacterial taxa having mean relative presence > 0.1% within a minimum of one cohort were encompassed; (h) Linear discriminant analysis (LDA) diagram of taxonomic differences between CO- and CB- cohort rabbits depending on LEfSe analysis. Species having major variations regarding presence with an LDA score > 3.0.Histogram bar reflects LDA scoring; (i) Cladogram demonstrates micro-organism-based populations that exhibited major variations across both cohorts. Red and green within phylogenetic-tree reflect micro-organism-based populations having pivotal parts within CB and CO cohorts respectively; (J) Heat map of Spearman correlation analysis results between bacterial genera and serum hormonal levels of rabbits. * p < 0.05; ^∗∗ p < 0.01, CO, control; CB, Can of beans.

Figure 4. Model of cecal microbiota variations and their effects on host physiology under enriched environments.