Resilience of Faecal Microbiota in Stabled Thoroughbred Horses Following Abrupt Dietary Transition between Freshly Cut Pasture and Three Forage-Based Diets

The management of competition horses in New Zealand often involves rotations of short periods of stall confinement and concentrate feeding, with periods of time at pasture. Under these systems, horses may undergo abrupt dietary changes, with the incorporation of grains or concentrate feeds to the diet to meet performance needs, or sudden changes in the type of forage fed in response to a lack of fresh or conserved forage. Abrupt changes in dietary management are a risk factor for gastrointestinal (GI) disturbances, potentially due to the negative effects observed on the population of GI microbiota. In the present study, the faecal microbiota of horses was investigated to determine how quickly the bacterial communities; (1) responded to dietary change, and (2) stabilised following abrupt dietary transition. Six Thoroughbred mares were stabled for six weeks, consuming freshly cut pasture (weeks 1, 3 and 5), before being abruptly transitioned to conserved forage-based diets, both offered ad libitum. Intestinal markers were administered to measure digesta transit time immediately before each diet change. The conserved forage-based diets were fed according to a 3 × 3 Latin square design (weeks 2, 4 and 6), and comprised a chopped ensiled forage fed exclusively (Diet FE) or with whole oats (Diet FE + O), and perennial ryegrass hay fed with whole oats (Diet H + O). Faecal samples were collected at regular intervals from each horse following the diet changes. High throughput 16S rRNA gene sequencing was used to evaluate the faecal microbiota. There were significant differences in alpha diversity across diets (p < 0.001), and a significant effect of diet on the beta diversity (ANOSIM, p = 0.001), with clustering of samples observed by diet group. There were differences in the bacterial phyla across diets (p < 0.003), with the highest relative abundances observed for Firmicutes (62–64%) in the two diets containing chopped ensiled forage, Bacteroidetes (32–38%) in the pasture diets, and Spirochaetes (17%) in the diet containing hay. Major changes in relative abundances of faecal bacteria appeared to correspond with the cumulative percentage of intestinal markers retrieved in the faeces as the increasing amounts of digesta from each new diet transited the animals. A stable faecal microbiota profile was observed in the samples from 96 h after abrupt transition to the treatment diets containing ensiled chopped forage. The present study confirmed that the diversity and community structure of the faecal bacteria in horses is diet-specific and resilient following dietary transition and emphasised the need to have modern horse feeding management that reflects the ecological niche, particularly by incorporating large proportions of forage into equine diets

Seasonal variation in the faecal microbiota of mature adult horses maintained on pasture in New Zealand

10.3390/ani11082300Animals118230

Rarefaction curves for microbial communities in faecal samples of forage-fed horses.

<p>The rarefaction curves show the mean number (with standard deviation) of observed species against the depth of sequencing of bacterial (-ο-), archaeal (-Δ-) and ciliate protozoal (--) communities in the equine faeces sampled in the study (n = 72 faecal samples). Multiple rarefactions were calculated from the OTU tables obtained for each of the three microbial groups representing 71 out of 72 samples for bacteria (minimum of 1,000 sequence reads/sample), 70 out of 72 samples for archaea (minimum of 320 sequence reads/sample) and 26 out of 37 samples for ciliate protozoa (minimum of 250 sequence reads/sample).</p

Faecal Microbiota of Forage-Fed Horses in New Zealand and the Population Dynamics of Microbial Communities following Dietary Change

<div><p>The effects of abrupt dietary transition on the faecal microbiota of forage-fed horses over a 3-week period were investigated. Yearling Thoroughbred fillies reared as a cohort were exclusively fed on either an ensiled conserved forage-grain diet (“Group A”; n = 6) or pasture (“Group B”; n = 6) for three weeks prior to the study. After the Day 0 faecal samples were collected, horses of Group A were abruptly transitioned to pasture. Both groups continued to graze similar pasture for three weeks, with faecal samples collected at 4-day intervals. DNA was isolated from the faeces and microbial 16S and 18S rRNA gene amplicons were generated and analysed by pyrosequencing. The faecal bacterial communities of both groups of horses were highly diverse (Simpson’s index of diversity >0.8), with differences between the two groups on Day 0 (<i>P</i><0.017 adjusted for multiple comparisons). There were differences between Groups A and B in the relative abundances of four genera, BF311 (family Bacteroidaceae; <i>P</i> = 0.003), CF231 (family Paraprevotellaceae; <i>P</i> = 0.004), and currently unclassified members within the order Clostridiales (<i>P</i> = 0.003) and within the family Lachnospiraceae (<i>P</i> = 0.006). The bacterial community of Group A horses became similar to Group B within four days of feeding on pasture, whereas the structure of the archaeal community remained constant pre- and post-dietary change. The community structure of the faecal microbiota (bacteria, archaea and ciliate protozoa) of pasture-fed horses was also identified. The initial differences observed appeared to be linked to recent dietary history, with the bacterial community of the forage-fed horses responding rapidly to abrupt dietary change.</p></div

Comparison of the bacterial community structure at multiple levels.

<p>The median relative abundances of the bacterial phyla in the faeces of Group A and B horses, on Days 0 and 4, are illustrated in the figure, panel (A). Panel (B) shows the relative abundances of the bacterial families, and panel (C) shows the relative abundances of bacterial genera present in the faeces of Group A and B horses, on Days 0 and 4. The stacked bar graph is presented in ascending order of the median relative abundances of bacterial taxa for Group A horses, and the legends show the most dominant taxa in each graph (>15% median relative abundance for the phylum level and >8% for the family and genus levels).</p

Metrics of data generated by 454 GS FLX Titanium pyrosequencing of 16S and 18S rRNA gene amplicons from microbial groups present in 72 equine faecal samples.

<p>*Not applicable (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112846#s2" target="_blank">Materials and Methods</a> section).</p>§<p>Samples with low number of sequences were excluded from the microbial libraries (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112846#s2" target="_blank">Materials and Methods</a> section).</p><p>Metrics of data generated by 454 GS FLX Titanium pyrosequencing of 16S and 18S rRNA gene amplicons from microbial groups present in 72 equine faecal samples.</p

Comparison of the relative abundances of bacterial genera in the faeces of Group A (fed Diet F, n = 6) and Group B (fed Diet P, n = 6) horses on Day 0.

<p>The table lists bacterial genera that were present at relative abundances of ≥4% in both Groups A and B, and certain genera (present at <4% relative abundance) that were different between Groups A and B on Day 0. The taxonomic ranks are listed from Phylum to Genus in descending order of relative abundances for Group A.</p>a<p>IQR–Interquartile range.</p>b<p>Level of statistical significance after Bonferroni adjustment for multiple comparisons <i>P</i> = 0.001.</p><p>*Differences between Groups A and B.</p><p>Comparison of the relative abundances of bacterial genera in the faeces of Group A (fed Diet F, n = 6) and Group B (fed Diet P, n = 6) horses on Day 0.</p